Generation and isolation of antigen-specific T cells

ABSTRACT

The present invention relates generally to methods for generating, expanding, and isolating antigen-specific T cells. Compositions of antigen-specific T cells activated and expanded by the methods herein are further provided.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention relates generally to methods forgenerating, isolating, and expanding antigen-specific T cells. Thepresent invention also relates to compositions of antigen-specific Tcells.

[0003] 2. Description of Related Art

[0004] The identification of antigens recognized by T cells in a varietyof cancers and infectious diseases has contributed significantly to theinterest in the use of antigen-specific immunotherapy for the treatmentof malignancies and infectious diseases. Adoptive therapy usingantigen-specific T cells represents a conceptually attractive strategyby providing a means to manipulate the specificity, phenotype andmagnitude of the intended immune response. Methods to routinely andreproducibly expand antigen-specific T cell clones for use in clinicaltrials of adoptive therapy would be desirable. Current technologies forgenerating therapeutic doses of antigen-specific T cells remain limitedand could be improved by simplifying the manufacturing process whilemaintaining or perhaps improving the function of the infused T cells.

[0005] The various techniques available for expanding human T-cells haverelied primarily on the use of accessory cells (primarily antigenpresenting cells (APC)) and/or exogenous growth factors, such asinterleukin-2 (IL-2). IL-2 has been used together with an anti-CD3antibody to stimulate T-cell proliferation, predominantly expanding theCD8⁺ subpopulation of T-cells. Both APC signals are thought to berequired for optimal T-cell activation, expansion, and long-termsurvival of the T-cells upon re-infusion. The requirement forMHC-matched APCs as accessory cells presents a significant problem forlong-term culture systems because APCs are relatively short-lived.Therefore, in a long-term culture system, APCs must be continuallyobtained from a source and replenished. The necessity for a renewablesupply of accessory cells is problematic for treatment ofimmunodeficiencies in which accessory cells are affected. In addition,when treating viral infection, if accessory cells carry the virus, thecells may contaminate the entire T-cell population during long-termculture.

[0006] Further, similar syStems require vaccination with antigen (e.g.tumor/viral antigen), pulsing of antigen-presenting cells with antigensfollowed by infusion of cells. Expansion of antigen-specific T cells togenerate large numbers of antigen-specific T cells often requires laborintensive and expensive cloning, and/or multiple rounds ofactivation/expansion to achieve therapeutically relevant T cell numbers.

[0007] Therefore, there is a need in the art for improved methods toroutinely and reproducibly expand antigen-specific T cell clones for usein clinical trials of adoptive therapy and for a simplifiedmanufacturing process that maintains or even improves the function ofthe antigen-specific T cells.

[0008] The present invention provides methods to generate an increasednumber of highly responsive antigen-specific T cells that have surfacereceptor and cytokine production characteristics that are more moredesirable than other expansion methods. The instant invention does notrequire knowledge of a particular antigen (although known antigens canbe used in the context of this invention) and provides for a single, ordouble, round of expansion to achieve a therapeutically relevant dose ofantigen-specific T cells, both of the CD4 and CD8 lineage (and eithermay be selected if desired).

SUMMARY OF THE INVENTION

[0009] Generally, the present invention relates to methods foractivating, stimulating and isolating antigen-specific T cells. Thepresent invention also relates to compositions of antigen-specific Tcells and methods of their use in the treatment and prevention ofcancer, infectious diseases, autoimmune diseases, immune disfunctionrelated to aging, or any other disease state where antigen-specific Tcells are desired for treatment.

[0010] In one aspect of the present invention, a method for expanding apopulation of antigen-specific T cells is provided, comprisingcontacting a population of cells wherein at least a portion thereofcomprises antigen-specific T cells, with a surface, wherein said surfacehas attached thereto a first agent and a second agent, wherein saidfirst agent ligates a CD3/TCR complex on said T cells and said secondagent ligates an accessory molecule on said T cells, and wherein saidligation by said first and second agent of said T cells inducesproliferation of antigen-specific T cells and wherein said surface ispresent in a ratio of surface to T cells of 1:2 or less. In certainembodiments the ratio of surface to T cells is between about 1:1 andabout 1:50 and any ratio therebetween. In certain embodiments the ratioof surface to T cells is from about 1:2, 1:2.5, 1:5, 1:10, 1:25, 1:50,1:75, 1:100, or lower. In one embodiment, the surface includes but isnot limited to paramagnetic beads, lipids, and cell surfaces. In certainembodiments, the surface comprises paramagnetic beads conjugated to oneor more antibodies. In certain embodiments, the surface can have 1, 2,3, 4, or more antibodies or natural ligands conjugated thereto.

[0011] Another aspect of the present invention provides a method forgenerating antigen-specific T cells comprising exposing a firstpopulation of cells wherein at least a portion thereof comprises antigenpresenting cells (APC) to a surface wherein said surface has antigenattached thereto, such that said surface with antigen attached theretois ingested by said APC; exposing a second population of cells whereinat least a portion thereof comprises T cells to the population of cellsin part (a); thereby generating antigen-specific T cells. Antigen may beattached or coupled to, or integrated into a surface by a variety ofmethods known and available in the art and described herein. In oneembodiment, the antigen is crosslinked to said surface. In a furtherembodiment, the attachment to said surface is by covalent ornoncovalent, electrostatic, or hydrophobic and may be accomplished by avariety of attachment means, including for example, chemical,mechanical, enzymatic, electrostatic, or other means whereby theantigen(s) is capable of stimulating the cells. For example, theantibody to an antigen first may be attached to a surface, or avidin orstreptavidin may be attached to the surface for binding to abiotinylated antigen. The antibody to the ligand may be attached to thesurface via an anti-idiotype antibody. Another example includes usingprotein A or protein G, or other non-specific antibody bindingmolecules, attached to surfaces to bind an antibody. Alternatively,antigen may be attached to the surface by chemical means, such ascross-linking to the surface, using commercially available cross-linkingreagents (Pierce, Rockford, Ill.) or other means. In certainembodiments, antigens are covalently bound to the surface. Further, inone embodiment, commercially available tosyl-activated DYNABEADS™ orDYNABEADS™ with epoxy-surface reactive groups are incubated with thepolypeptide antigen of interest according to the manufacturer'sinstructions. Briefly, such conditions typically involve incubation in aphosphate buffer from pH 4 to pH 9.5 at temperatures ranging from 4 to37 degrees C.

[0012] In one embodiment, the APC are in direct contact with theantigen-specific T cells. In a further embodiment, the APC that are indirect contact with said antigen-specific T cells are isolated byexposing said APC to a magnetic field, wherein said surface comprises aparamagnetic, magnetic, or magnetizable component. In anotherembodiment, the antigen-specific T cells are expanded by exposing said Tcells to a surface wherein said surface has attached thereto a firstagent that ligates a first T cell surface moiety of a T cell, and thesame or a second surface has attached thereto a second agent thatligates a second moiety of said T cell, wherein said ligation by thefirst and second agent induces proliferation (expansion) of saidantigen-specific T cells. In certain embodiments, at least one agent isan antibody or an antibody fragment. In other embodiments, the firstagent is an antibody or a fragment thereof, and the second agent is anantibody or a fragment thereof. In yet another embodiment, the first andthe second agents are different antibodies. In certain embodiments, thefirst agent is an anti-CD3 antibody, an anti-CD2 antibody, or anantibody fragment of an anti-CD3 or anti-CD2 antibody and the second thesecond agent is an anti-CD28 antibody or antibody fragment thereof. Inanother embodiment, the first agent is an anti-CD3 antibody and thesecond agent is an anti-CD28 antibody. In further embodiments, theanti-CD3 antibody and the anti-CD28 antibody are present at a ratio ofabout 1:1 to about 1:100. In a further embodiment, the antigen-specificT cells are expanded by exposing said antigen-specific T cells to amitogen, such as phytohemagglutinin (PHA), phorbol myristate acetate(PMA) and ionomycin, lipopolysaccharide (LPS), and superantigen.

[0013] In a further embodiment, the antigen of the present inventionincludes but is not limited to protein, glycoprotein, peptides,antibody/antigen complexes, whole tumor or virus-infected cells, fixedtumor or virus-infected cells, heat-killed tumor or virus-infectedcells, tumor lysate, virus lysate, non-soluble cell debris, apoptoticbodies, necrotic cells, whole tumor cells from a tumor or a cell linethat have been treated such that they are unable to continue dividing,allogeneic cells that have been treated such that they are unable tocontinue dividing, irradiated tumor cells, irradiated allogeneic cells,natural or synthetic complex carbohydrates, lipoproteins,lipopolysaccharides, transformed cells or cell line, transfected cellsor cell line, transduced cells or cell line, and virally infected cellsor cell line. In certain embodiments, antigen is attached to saidsurface by an antibody/ligand interaction. An antibody/ligandinteraction includes but is not limited to an interaction between anantibody/ligand pair selected from the group consisting of anti-MART-1antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1 antibody/PR1, anti-PR3 antibody /PR3, anti-tyrosinase antibody/tyrosinaseantigen, anti-MAGE-1 antibody/MAGE-1 antigen, anti-MUC-1 antibody/MUC-1antigen, anti-α-fetoprotein antibody/α-fetoprotein antigen, anti-Her2Neuantibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza HAantibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis Cantibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA 3Bantigen, and anti-human Ig heavy and lignt chains/Ig from a myelomacancer patient, and anti-human Ig heavy and lignt chains/Ig from a CLLcancer patient. In certain embodiments, the antigen is chemicallyattached to a surface. In one embodiment, the attachment of said antigento said surface comprises a biotin-avidin interaction. In a furtherembodiment, the population of cells wherein at least a portion thereofcomprises APC is derived from a source selected from the groupconsisting of a leukapheresis product, peripheral blood, lymph node,tonsil, thymus, tissue biopsy, tumor, spleen, bone marrow, cord blood,CD34⁺ cells, monocytes, and adherent cells.

[0014] Another aspect of the present invention provides a method forgenerating and expanding antigen-specific T cells comprising exposing afirst population of cells wherein at least a portion thereof comprisesantigen presenting cells to antigen such that said antigen is taken upby said APC; exposing a second population of cells wherein at least aportion thereof comprises T cells to the population of cells in part(a); thereby generating antigen-specific T cells; and exposing saidantigen-specific T cells of part (b) to a surface wherein said surfacehas attached thereto a first agent that ligates a first T cell surfacemoiety of a T cell, and the same or a second surface has attachedthereto a second agent that ligates a second moiety of said T cell,wherein said ligation by the first and second agent inducesproliferation (expansion) of said antigen-specific T cells. In certainembodiments, at least one agent is an antibody or an antibody fragment.In other embodiments, the first agent is an antibody or a fragmentthereof, and the second agent is an antibody or a fragment thereof. Inyet another embodiment, the first and the second agents are differentantibodies. In certain embodiments, the first agent is an anti-CD3antibody, an anti-CD2 antibody, or an antibody fragment of an anti-CD3or anti-CD2 antibody and the second the second agent is an anti-CD28antibody or antibody fragment thereof. In another embodiment, the firstagent is an anti-CD3 antibody and the second agent is an anti-CD28antibody. In further embodiments, the anti-CD3 antibody and theanti-CD28 antibody are present at a ratio of about 1:1 to about 1:100.In one embodiment said antigen-specific T cells are isolated bycontacting said T cells with antibodies specific for T cell activationmarkers. In another embodiment said antibodies are selected from thegroup consisting of anti-CD25, anti-CD54, anti-CD69, anti-CD38,anti-CD45RO, anti-CD49d, anti-CD40L, anti-CD137, anti-CD62L, andanti-CD134.

[0015] A further aspect of the present invention provides a populationof antigen-specific T cells generated according to any one of themethods described herein.

[0016] An additional aspect of this invention is a compositioncomprising the antigen-specific T cells according to any of the methodsdescribed herein and a pharmaceutically acceptable excipient.

[0017] A further aspect of the present invention provides methods forstimulating an immune response in a mammal comprising, administering tothe mammal compositions comprising the antigen-specific T cells of thepresent invention.

[0018] An additional aspect of the invention provides for reducing thepresence of cancer cells in a mammal comprising, exposing the cancercells to the compositions comprising antigen-specific T cells. In oneembodiment, the cancer cells are from a cancer selected from the groupconsisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease,leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer,prostate cancer, colo-rectal cancer, kidney cancer, renal cellcarcinoma, pancreatic cancer, esophageal cancer, brain cancer, lungcancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma,acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia(CLL).

[0019] One aspect of the present invention provides a method forinhibiting the development of a cancer in a mammal, comprisingadministering to the mammal the composition comprising antigen-specificT cells fo the present invention. In certain embodiments, the cancercells are from a cancer selected from the group consisting of melanoma,non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectalcancer, kidney cancer, renal cell carcinoma, pancreatic cancer,esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervicalcancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),and chronic lymphocytic leukemia (CLL).

[0020] A further aspect of the present invention provides a method forameliorating an immune response dysfunction in a mammal comprisingadministering to the mammal the compositions comprising antigen-specificT cells generated using any one of the methods described herein.

[0021] Yet another aspect of the invention provides a method forreducing the presence of an infectious organism in a mammal comprising,administering to the mammal a composition comprising antigen-specific Tcells generated using any one of the methods described herein. Withinthis context, an infectious organism can include but is not limited to avirus, a single-stranded RNA virus, a single-stranded DNA virus, adouble-stranded DNA virus, Human Immunodeficiency Virus (HIV), HepatitisA, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus(HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, abacterium, M. tuberculosis, Pneumocystis carinii, Candida, Aspergillus.

[0022] An additional aspect of the present invention provides a methodfor inhibiting the development of an infectious disease in a mammal,comprising administering to the mammal the compositions comprisingantigen-specific T cells generated using any one of the methodsdescribed herein. In this regard an infectious disease can be caused byan infectious organism including but not limited to a virus, an RNAvirus, a DNA virus, Human Immunodeficiency Virus (HIV), Hepatitis A, B,or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus (HPV),Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, abacterium, M. tuberculosis, Pneumocystis carinii, Candida, Aspergillus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a photograph showing the tight association ofantigen-specific T cells and bead-loaded antigen presenting cells (APC)post magnetic separation.

[0024]FIG. 2 is a plot showing upregulation of CD25 in re-stimulatedmemory CD8 CMV tetramer+ T cells expanded ex vivo. Panel A is a negativecontrol from an HLA-A2+, CMV− donor. Panel B is a negative controlshowing uncoated bead stimulation from an HLA-A2+, CMV+ donor. Panel Cshows CMV antigen-coated bead stimulation of cells from an HLA-A2+, CMV+donor.

[0025]FIG. 3 is a plot showing the effect of varying bead:cell ratio onexpansion or deletion of CMV-specific T cells.

[0026]FIG. 4 panels A and B is a bar graph showing the effect on T cellexpansion of sequential bead addition at varying bead:cell ratios atvarying times during cutlure. Panel A shows a comparison of total T cellexpansion over 15 days, comparing standard static cuture (beads at day 0at either 1:2.5 or 1:5 bead to cell ratio) or additional beads added atday 5, 7, or 9 at 1:10, 1:25, 1:50 or 1:100 bead to cell ratios. Panel Bshows CMV-specific T cell expansion under the same experimentalconditions as Panel A.

[0027]FIG. 5 is a graph showing the effect on T cell expansion of lowbead:T cell ratio and sequential addition of beads on Melanomagp100(M)-specific T cells.

[0028]FIG. 6 is a graph depicting the fold increase of T-cells over timefollowing stimulation with anti-CD3 and anti-CD28 co-immobilized beadswith varying ratios of anti-CD3:CD28 antibodies attached thereto.

[0029]FIG. 7 is a graph depicting the fold increase of CMVpp65-specificT-cells over time following stimulation with anti-CD3 and anti-CD28co-immobilized beads with varying ratios of anti-CD3:CD28 antibodiesattached thereto.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

[0031] The term “biocompatible”, as used herein, refers to the propertyof being predominantly non-toxic to living cells.

[0032] The term “stimulation”, as used herein, refers to a primaryresponse induced by ligation of a cell surface moiety. For example, inthe context of receptors, such stimulation entails the ligation of areceptor and a subsequent signal transduction event. With respect tostimulation of a T-cell, such stimulation refers to the ligation of aT-cell surface moiety that in one embodiment subsequently induces asignal transduction event, such as binding the TCR/CD3 complex. Further,the stimulation event may activate a cell and upregulate or downregulateexpression or secretion of a molecule, such as downregulation of TGF-β.Thus, ligation of cell surface moieties, even in the absence of a directsignal transduction event, may result in the reorganization ofcytoskeletal structures, or in the coalescing of cell surface moieties,each of which could serve to enhance, modify, or alter subsequent cellresponses.

[0033] The term “activation”, as used herein, refers to the state of acell following sufficient cell surface moiety ligation to induce anoticeable biochemical or morphological change. Within the context ofT-cells, such activation, refers to the state of a T-cell that has beensufficiently stimulated to induce cellular proliferation. Activation ofa T-cell may also induce cytokine production and performance ofregulatory or cytolytic effector functions. Within the context of othercells, this term infers either up or down regulation of a particularphysico-chemical process.

[0034] The term “target cell”, as used herein, refers to any cell thatis intended to be stimulated by cell surface moiety ligation.

[0035] An “antibody”, as used herein, includes both polyclonal andmonoclonal antibodies; primatized (e.g., humanized); murine;mouse-human; mouse-primate; and chimeric; and may be an intact molecule,a fragment thereof (such as scFv, Fv, Fd, Fab, Fab′ and F(ab)′₂fragments), or multimers or aggregates of intact molecules and/orfragments; and may occur in nature or be produced, e.g., byimmunization, synthesis or genetic engineering; an “antibody fragment,”as used herein, refers to fragments, derived from or related to anantibody, which bind antigen and which in some embodiments may bederivatized to exhibit structural features that facilitate clearance anduptake, e.g., by the incorporation of galactose residues. This includes,e.g., F(ab), F(ab)′₂, scFv, light chain variable region (V_(L)), heavychain variable region (V_(H)), and combinations thereof.

[0036] The term “protein”, as used herein, includes proteins,polypeptides and peptides; and may be an intact molecule, a fragmentthereof, or multimers or aggregates of intact molecules and/orfragments; and may occur in nature or be produced, e.g., by synthesis(including chemical and/or enzymatic) or genetic engineering.

[0037] The term “agent”, “ligand”, or “agent that binds a cell surfacemoiety”, as used herein, refers to a molecule that binds to a definedpopulation of cells. The agent may bind any cell surface moiety, such asa receptor, an antigenic determinant, or other binding site present onthe target cell population. The agent may be a protein, peptide,antibody and antibody fragments thereof, fusion proteins, syntheticmolecule, an organic molecule (e.g., a small molecule), or the like.Within the specification and in the context of T-cell stimulation,antibodies are used as a prototypical example of such an agent.

[0038] The terms “agent that binds a cell surface moiety” and “cellsurface moiety”, as used herein, are used in the context of aligand/anti-ligand pair. Accordingly, these molecules should be viewedas a complementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity (an affinityconstant, K_(a), of about 10⁶ M⁻¹ or tighter).

[0039] “Antigen-presenting cell (APC)”, as used herein, refers to thosecells that normally initiate the responses of naïve and/or memory Tcells to antigen. In this regard, APC refers to any cell capable ofantigen presentation. APCs include, but are not limited to, dendriticcells, monocytes, macrophages, and B cells. An APC may express highlevels of MHC class II, ICAM-1 and B7-2.

[0040] A “co-stimulatory signal”, as used herein, refers to a signal,which in combination with a primary signal, such as TCR/CD3 ligation,leads to T-cell proliferation.

[0041] A “ligand/anti-ligand pair”, as used herein, refers to acomplementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity (an affinityconstant, K_(a), of at least about 10⁶ M⁻¹,). The skilled artisan wouldunderstand that this affinity is illustrative only and that affinityconstants of the ligand/anti-ligand pairs useful in the context of thepresent invention might be lower or in some cases higher. For example,in the case of biotin/streptavidin, the streptavidin on-rate iscomparable to that of monomeric avidin while its off-rate is seven timeslower. The dissociation constant was determined to be 1.3×10(−8)M.Exemplary ligand/anti-ligand pairs enzyme/inhibitor, hapten/antibody,lectin/carbohydrate, ligand/receptor, and biotin/avidin or streptavidin.Within the context of the present invention specification receptors andother cell surface moieties are anti-ligands, while agents (e.g.,antibodies and antibody fragments) reactive therewith are consideredligands.

[0042] “Separation”, as used herein, includes any means of substantiallypurifying one component from another (e.g., by filtration, magneticattraction, etc.).

[0043] “Quiescent”, as used herein, refers to a cell state wherein thecell is not actively proliferating.

[0044] A “surface”, as used herein, refers to any surface capable ofhaving an agent attached thereto and includes, without limitation,metals, glass, plastics, co-polymers, colloids, lipids, cell surfaces,and the like. Essentially any surface that is capable of retaining anagent bound or attached thereto. A prototypical example of a surfaceused herein, is a particle such as a bead. As such, the terms “surface”and “particle” are used herein interchangeably.

[0045] “Immune response or responsiveness” as used herein, refers toactivation of cells of the immune system, including but not limited to,T-cells, such that a particular effector function(s) of a particularcell is induced. Effector functions may include, but are not limited to,proliferation, secretion of cytokines, secretion of antibodies,expression of regulatory and/or adhesion molecules, and the ability toinduce cytolysis.

[0046] “Stimulating an immune response” as used herein, refers to anystimulation such that activation and induction of effector functions ofcells of the immune system are achieved.

[0047] “Immune response dysfunction” as used herein, refers to theinappropriate activation and/or proliferation, or lack thereof, of cellsof the immune system, and/or the inappropriate secretion, or lackthereof, of cytokines, and/or the inappropriate or inadequate inductionof other effector functions of cells of the immune system, such asexpression of regulatory, adhesion, and/or homing receptors, and theinduction of cytolysis.

[0048] The terms “preventing” or “inhibiting” the development of acancer or cancer cells” as used herein, refers to the occurrence of thecancer being prevented or the onset of the cancer being delayed.

[0049] The term “treating” or “reducing the presence of a cancer orcancer cells” as used herein, means that the cancer growth is inhibited,which is reflected by, e.g., tumor volume or numbers of malignant cells.Tumor volume may be determined by various known procedures, e.g.,obtaining two dimensional measurements with a dial caliper.

[0050] “Preventing or inhibiting the development of an infectiousdisease” as used herein, means the occurrence of the infectious diseaseis prevented or the onset of the infectious disease is delayed, or thespread of an existing infection is reversed.

[0051] “Ameliorate” as used herein, is defined as: to make better;improve (The American Heritage College Dictionary, 3^(rd) Edition,Houghton Mifflin Company, 2000).

[0052] “Particles” as used herein, may include a colloidal particle, amicrosphere, nanoparticle, a bead, or the like. In the variousembodiments, commercially available surfaces, such as beads or otherparticles, are useful (e.g., Miltenyi Particles, Miltenyi Biotec,Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADS™,Dynal Inc., Oslo, Norway; PURABEADS™, Prometic Biosciences, magneticbeads from Immunicon, Huntingdon Valley, Pa., microspheres from BangsLaboratories, Inc., Fishers, Ind.).

[0053] “Paramagnetic particles” as used herein, refer to particles, asdefined above, that localize in response to a magnetic field.

[0054] “Antigen” as used herein, refers to any molecule 1) capable ofbeing specifically recognized, either in its entirety or fragmentsthereof, and bound by the “idotypic” portion (antigen-binding region) ofa mAb or its derviative; 2) containing peptide sequences which can bebound by MHC and then, in the context of MHC presentation, canspecifically engage its cognate T cell antigen receptor.

[0055] To “load” an APC with antigen, as used herein, refers to exposingan APC to antigen or antigenic peptide for a period of time sufficientfor the APC to uptake, process, and present the antigen, bound by MHCmolecules, to T cells. In some cases, the antigen, especially peptide,can be bound by MHC molecules and presented to T cells without beingtaken up and processed by the APC.

[0056] The term “animal” or “mammal” as used herein, encompasses allmammals, including humans. Preferably, the animal of the presentinvention is a human subject.

[0057] The term “exposing” as used herein, refers to bringing into thestate or condition of immediate proximity or direct contact.

[0058] The term “lysate” as used herein, refers to the supernatant andnon-soluble cell debris resulting from lysis of cells. A skilled artisanwill recognize that any number of lysis buffers known in the art may beused (see for example Current Protocols in Immunology, John Wiley &Sons, New York. N.Y.). Cell lysis may also be carried out by freeze-thawprocedures or other means (e.g. sonication, etc.).

[0059] The term “apoptotic body” as used herein, is defined as thesmaller, intact, membrane-bound fragments that result from apoptoticcells.

[0060] The term “proliferation” as used herein, means to grow ormultiply by producing new cells.

[0061] The term “infectious disease” as used herein, refers to anydisease that is caused by an infectious organism. Infectious organismsmay comprise viruses, (e.g., RNA viruses, DNA viruses, humanimmunodeficiency virus (HIV), hepatitis A, B, and C virus, herpessimplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr virus (EBV),human papilloma virus (HPV)), parasites (e.g., protozoan and metazoanpathogens such as Plasmodia species, Leishmania species, Schistosomaspecies, Trypanosoma species), bacteria (e.g., Mycobacteria, inparticular, M. tuberculosis, Salmonella, Streptococci, E. coli,Staphylococci), fungi (e.g., Candida species, Aspergillus species),Pneumocystis carinii, and prions (known prions infect animals to causescrapie, a transmissible, degenerative disease of the nervous system ofsheep and goats, as well as bovine spongiform encephalopathy (BSE), or“mad cow disease”, and feline spongiform encephalopathy of cats. Fourprion diseases known to affect humans are (1) kuru, (2)Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-ScheinkerDisease (GSS), and (4) fatal familial insomnia (FFI)). As used herein“prion” includes all forms of prions causing all or any of thesediseases or others in any animals used—and in particular in humans anddomesticated farm animals.

[0062] Sources of T Cells

[0063] T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, thymus, tissue biopsy,tumor, lymph node tissue, gut associated lymphoid tissue, mucosaassociated lymphoid tissue, spleen tissue, or any other lymphoid tissue,and tumors. T cells can be obtained from T cell lines and fromautologous or allogeneic sources. T cells may also be obtained from axenogeneic source, for example, from mouse, rat, non-human primate, andpig.

[0064] Preferably, cells from the circulating blood of an individual areobtained by apheresis or leukapheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis orleukapheresis may be washed to remove the plasma fraction and to placethe cells in an appropriate buffer or media for subsequent processingsteps. In one embodiment of the invention, the cells are washed withphosphate buffered saline (PBS). In an alternative embodiment, the washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations. As those of ordinary skill in the art wouldreadily appreciate a washing step may be accomplished by methods knownto those in the art, such as by using a semi-automated “flow-through”centrifuge (for example, the Cobe 2991 cell processor, Baxter) accordingto the manufacturer's instructions. After washing, the cells may beresuspended in a variety of biocompatible buffers, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

[0065] In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and by centrifugation througha PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28⁺,CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, can be further isolated bypositive or negative selection techniques. For example, CD3⁺, CD28⁺ Tcells can be positively selected using CD3/CD28 conjugated magneticbeads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). In one aspectof the present invention, enrichment of a T cell population by negativeselection can be accomplished with a combination of antibodies directedto surface markers unique to the negatively selected cells. A preferredmethod is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4⁺cells by negativeselection, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0066] Another method for preparing T cells for stimulation is to freezethe cells after the washing step, which does not require themonocyte-removal step. Wishing not to be bound by theory, the freeze andsubsequent thaw step provides a more uniform product by removinggranulocytes and, to some extent, monocytes in the cell population.After the washing step that removes plasma and platelets, the cells maybe suspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serum albumin(HSA), or other suitable cell freezing media. This is then diluted 1:1with media so that the final concentration of DMSO and HSA are 10% and4%, respectively. The cells are then frozen to −80° C. at a rate of 1°per minute and stored in the vapor phase of a liquid nitrogen storagetank.

[0067] Sources of Antigen-Presenting Cells (APC)

[0068] The source of antigen-presenting cell (APC) is typically a tissuesource comprising APC or APC precursors that are capable ofproliferating and maturing in vitro into professional APC (pAPC) whenloaded with antigen and/or treated with the necessary cytokines orfactors. “Professional APC” (pAPC) or “antigen-presenting cell” (APC),as used herein, refers to those cells that normally initiate theresponses of naïve and/or memory T cells to antigen. Professional APCsinclude, but are not limited to, DC, macrophages, and B cells. pAPC mayexpress high levels of MHC class II, ICAM-1 and B7-2. In one aspect, APCprecursor cells are capable of proliferating and maturing in vitro intodendritic cells (DC). While many tissue sources may be used, typicaltissue sources comprise spleen, thymus, tissue biopsy, tumor, afferentlymph, lymph nodes, bone marrow, apheresis or leukapheresis product,and/or peripheral blood. In certain embodiments, apheresis product, bonemarrow and peripheral blood are preferred sources. Fetal tissue, fetalor umbilical cord blood, which is also rich in growth factors may alsobe used as a source of blood for obtaining APC and/or precursor APC.Exemplary precursor cells may be, but are not limited to, embryonic stemcells, CD34⁺ cells, monocyte progenitors, monocytes, and pre-B cells.

[0069] Further, according to one aspect of the present invention, APCmay be derived from precursor cells comprising monocytes or CD34⁺ cells.

[0070] In one aspect of the present invention, the source of APC and/orprecursor APC is an apheresis or leukapheresis product. Cells arecollected using apheresis procedures known in the art. See, for example,Bishop et al., Blood, vol. 83, No. 2, pp. 610-616 (1994). Briefly, cellsare collected using conventional devices, for example, a HaemoneticsModel V50 apheresis device (Haemonetics, Braintree, Mass.). Apheresisproduct typically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In another embodiment of the invention, the cells are washed withphosphate buffered saline (PBS). In an alternative embodiment, the washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations. As those of ordinary skill in the art wouldreadily appreciate a washing step may be accomplished by methods knownto those in the art, such as by using a semi-automated “flow-through”centrifuge (for example, the Cobe 2991 cell processor, Gambro BCT,Lakewood, Colo.) according to the manufacturer's instructions. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

[0071] When blood is used as a source of APC, blood leukocytes may beobtained using conventional methods that maintain their viability.According to one aspect of the invention, blood is diluted into medium(preferably RPMI) that may or may not contain heparin (about 100 U/ml)or other suitable anticoagulant. The volume of blood to medium is about1 to 1. Cells are concentrated by centrifugation of the blood in mediumat about 1000 rpm (150 g) at 4° C. Platelets and red blood cells aredepleted by resuspending the cells in any number of solutions known inthe art that will lyse erythrocytes, for example ammonium chloride. Forexample, the mixture may be medium and ammonium chloride (at a finalconcentration of about 0.839 percent) at about 1:1 by volume. Cells maybe concentrated by centrifugation and washed in the desired solutionuntil a population of leukocytes, substantially free of platelets andred blood cells, is obtained, typically about two times. Any isotonicsolution commonly used in tissue culture may be used as the medium forseparating blood leukocytes from platelets and red blood cells. Examplesof such isotonic solutions are phosphate buffered saline, Hanks balancedsalt solution, or complete growth media including for example RPMI 1640,DMEM, MEM, HAMS F-12, X-Vivo 15, or X-Vivo 20. APC and/or APC precursorcells may also purified by elutriation, using, for example, a BeckmanJ6ME centrifuge equipped with a J5.0 rotor and a 40 ml elutriationchamber.

[0072] In one embodiment of the present invention, isolation of APCand/or precursor APC is performed by preincubating ficolled whole bloodor apheresed peripheral blood with one or more varieties of irrelevantor non-antibody coupled paramagnetic particles (approx. 1 vial of beadsor 4×10⁹ beads to one batch of cells (typically from about 5×10⁸ toabout 2×10¹⁰ cells) for about 30 minutes to 2 hours at 22 to 37 degreesC., followed by magnetic removal of cells which have attached to orengulfed the paramagnetic particles. Such separation can be performedusing standard methods available in the art. For example, any magneticseparation methodology may be used including a variety of which arecommercially available, (e.g., DYNAL® Magnetic Particle Concentrator(DYNAL MPC®)). Assurance of isolation can be monitored by a variety ofmethodologies known to those of ordinary skill in the art, includingflow cytometric analysis of cells before and after said isolation.

[0073] APC obtained from treatment of the tissue source may be culturedto form a primary culture in an appropriate culture container or vesselin an appropriate culture medium. In certain embodiments, the culturemedium is supplemented with one or more cytokines. According to thepresent invention, the appropriate culture container or vessel may beany container with tissue culture compatible surface. Examples includevarious bags (e.g., Lifecell culture bags), flasks, roller bottles,petri dishes and multi-well containing plates made for use in tissueculture. Surfaces treated with a substance, for example collagen orpoly-L-lysine, or antibodies specific for a particular cell type topromote cell adhesion may also be used provided they allow for thedifferential attachment of cells as described below. Surfaces may bealso be chemically treated, for example by ionization. Cells are platedat an initial cell density from about 10⁵ to 10⁷ cells/cm². In oneaspect, cells are plated at 10⁶ cell s/cm².

[0074] In one embodiment, the primary cultures from the selected tissuesource are allowed to incubate at about 37° C. under standard tissueculture conditions of humidity, CO₂, and pH until a population of cellshas adhered to the substrate sufficiently to allow for the separation ofnonadherent cells. Some immature APC in blood initially are nonadherentto plastic, particularly immature DC, in contrast to monocytes, so thatthe precursors can be separated after overnight culture. Monocytes andfibroblasts are believed to comprise the majority of adherent cells andusually adhere to the substrate within about 30 minutes to about 24hours. In certain aspects, nonadherent cells are separated from adherentcells between about 1 to 16 hours. Nonadherent cells may be separated atabout 1 to 2 hours. Any method which does not dislodge significantquantities of adherent cells may be used to separate the adherent fromnonadherent cells. In certain aspects, the cells are dislodged by simpleshaking or pipetting. Pipetting is most preferred.

[0075] Adherent cells comprising precursor APC (e.g., monocytes)isolated according to the methods of the invention are allowed toincubate at about 37° C. under standard tissue culture conditions ofhumidity, CO₂, and pH until a population of cells has reached animmature APC stage. In certain aspects, according to the presentinvention, adherent cells are allowed to incubate for a period ofbetween 4 hours and 7 days. However, one of ordinary skill in the artwill readily appreciate that incubation times and conditions may vary.“Immature APC” as used herein, refers to an intermediate differentiationstate of an APC wherein the APC has the capacity to endocytose orphagocytose antigen, foreign bodies, necrotic and/or apoptosing tissueand/or cells. Immature APC may be CD14⁻ or CD14⁺depending on the originof the precursor cells. Immature APC may also express CD1a, CD40, CD86,CD54, and intermediate levels of MHC class II (levels of markerexpression on sample cells can be compared by flow cytometric analysisto levels of expression on MHC class II-negative cells and cells knownto express high levels of MHC class II). Immature APC typically do notexpress CCR7.

[0076] In certain aspects of the present invention, it is not necessaryto separate T cells from APC. For example, in one embodiment, PBMCcomprising APC and T cells can be exposed to antigen as described hereinand the resulting antigen-specific T cells further expanded as describedherein.

[0077] In certain aspects of the present invention, it is not requiredthat the APCs or the T cells described herein be derived from anautologous source. Thus, the APC and T cells can be obtained from amatched or unmatched donor, or from a cell line, a T cell line, or othercells grown in vitro. Methods for matching haplotypes are known in theart. Furthermore, the APC and T cells or supernatant therefrom may beobtained from a xenogeneic source, for example, mouse, rat, non-humanprimate, and porcine cells may be used.

[0078] Sources of Antigen

[0079] According to the present invention, the source of antigen may be,but is not limited to, protein, including glycoprotein, peptides(including pools of overlapping peptides), superantigens (e.g., SEA,SEB, TSST-1) antibody/antigen complexes, tumor lysate, viral lysate(e.g., CMV lysate and the like), non-soluble cell debris, apoptoticbodies, necrotic cells, whole cells which are live, fixed, irradiated,heat-killed or otherwise manipulated, whole tumor cells from a tumor ora cell line that have been treated such that they are unable to continuedividing, allogeneic cells that have been treated such that they areunable to continue dividing, irradiated tumor cells, irradiatedallogeneic cells, natural or synthetic complex carbohydrates,lipoproteins, lipopolysaccharides, RNA or a translation product of saidRNA, and DNA or a polypeptide encoded by said DNA. Non-transformed cellsare typically irradiated with gamma rays in the range of about 3000 to3600 rads, more preferably at about 3300 rads. Lymphoblastoid or tumorcell lines are typically irradiated with gamma rays in the range ofabout 6000 to 10,000 rads, more preferably at about 8000 rads. Necroticand apoptotic cells may be generated by physical, chemical, orbiological means. Necrotic cells are typically generated byfreeze-thawing, while apoptotic cells are generated using UVirradiation. UV and gamma irradiation, and freeze-thawing procedures arewell known in the art and are described, for example, in CurrentProtocols in Molecular Biology or Current Protocols in Immunology, JohnWiley & Sons, New York. N.Y.

[0080] Antigen source may also comprise non-transformed, transformed,transfected, or transduced cells or cell lines. Cells may betransformed, transfected, or transduced using any of a variety ofexpression or retroviral vectors known to those of ordinary skill in theart that may be employed to express recombinant antigens. Expression mayalso be achieved in any appropriate host cell that has been transformed,transfected, or transduced with an expression or retroviral vectorcontaining a DNA molecule encoding recombinant antigen(s). Any number oftransfection, transformation, and transduction protocols known to thosein the art may be used, for example those outlined in Current Protocolsin Molecular Biology, John Wiley & Sons, New York. N.Y., or in numerouskits available commercially (e.g., Invitrogen Life Technologies,Carlsbad, Calif.). In one embodiment of the present invention,recombinant vaccinia vectors and cells infected with said vaccinavectors, may be used as a source of antigen. Recombinant antigen mayinclude any number of defined tumor antigens described below.

[0081] According to certain methods of the invention, antigen maycomprise viral antigens such as CMV pp65, HIV pg120, and the like. Incertain embodiments, antigen may comprise defined tumor antigens such asthe melanoma antigen Melan-A (also referred to as melanoma antigenrecognized by T cells or MART-1), melanoma antigen-encoding genes 1, 2,and 3 (MAGE-1, -2, -3), melanoma GP100, carcinoembryonic antigen (CEA),the breast cancer angtigen, Her-2/Neu, serum prostate specific antigen(PSA), Wilm's Tumor (WT-1), PR1, PR3 (antigens implicated in thegraft-versus-leukemia (GVL) effect in chronic myeloid leukemia), mucinantigens, MUC-1, -2, -3, -4, B cell lymphoma idotypes, and the like. Theskilled artisan would appreciate that any tumor antigen would be usefulin the context of the present invention.

[0082] Activation of Antigen-Specific T Cells

[0083] One aspect of the present invention stems from the surprisingfinding that using different bead:cell ratios can lead to differentoutcomes with respect to expansion of antigen-specific T cells. Inparticular, bead:cell ratios can be varied to selectively expand ordelete antigen-specific (memory) T cells. In one embodiment, theparticular bead:cell ratio used selectively expands antigen-specific Tcells. Thus, in one embodiment of the present invention,antigen-specific T cells are activated by direct contact of a populationof cells wherein at least a portion thereof comprises T cells (e.g., aleukaphersis product from an individual, blood sample, tumor biopsy,etc.), with a surface, wherein said surface has attached thereto a firstagent that ligates a first T cell surface moiety of a T cell, and thesame or a second surface has attached thereto a second agent thatligates a second moiety of said T cell, wherein said ligation by thefirst and second agent induces proliferation (expansion) ofantigen-specific T cells present within the population of cells.

[0084] Without being bound by theory, it is thought that theantigen-specific T cells are sensitized to further stimulation. Thus,the key appears to be the strength of the T cell activation signal:selective expansion of memory T cells (antigen-specific T cells) occurswith “weak” signals while selective deletion of memory T cells occurswith “strong” signals. The quantity of the CD3/TCR (and CD28) receptorsthat are bound by ligands determines the signal strength. Thus,stimulation with high bead:cell ratios provides a high concentration ofstimulating antibody (i.e., “strong signal”), leading toover-stimulation of antigen-specific T cells, causing them to die,either by apoptosis or other mechanisms. Using lower bead:cell ratiosprovides a stimulation signal to antigen-specific T cells that does notover-stimulate, but rather induces rapid proliferation of these cells.

[0085] In one embodiment of the present invention, antigen-specific Tcells are activated by culturing T cells isolated as described hereinabove, with APC that have been loaded with antigen.

[0086] In another embodiment, suitable APC are plated in culture dishesand exposed to a source of antigen as described herein, in a sufficientamount and for a sufficient period of time to allow the antigen to bindand/or be taken up by the APC. In certain aspects, antigen is exposed tothe APC for a period of time between 24 hours and 4 days. In oneparticular embodiment, the antigen is exposed to the APC for 36, 48, or72 hours. In a further embodiment, the antigen is exposed to the APC for2.5, 3, 3.5, or 4 days. In certain embodiments, antigen may be exposedto the APC for periods longer than 4 days, for example 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days. The amount and time necessaryto achieve binding and uptake of the antigen by the APC may differdepending on the source and type of antigen and may be determined bythose of ordinary skill in the art by immunoassay or binding assay.Other methods known to those of skill in the art may be used to detectthe presence of antigen in the context of MHC on the APC following theirexposure to antigen.

[0087] In yet an additional embodiment, PBMC (e.g., from blood, aleukapheris product, etc.) from a subject are cultured directly in thepresence of antigen, as described herein, to load APC with the antigenand to activate/stimulate antigen-specific T cells present in the PBMC.In this regard, PBMC may be collected from an individual, contacted withan antigen of interest, such as a tumor antigen, or a viral lysate, etc.In this manner, the APC present in the PBMC are loaded with the antigen,which is then presented to the T cells present in the sample. In anadditional embodiment, the antigen-specific T cells of the presentinvention may be stimulated with peptide-MHC tetramers, see for exampleAltman, et al., Science 1998 Jun. 19; 280(5371):1821.

[0088] The APC of the present invention may be loaded with antigenthrough genetic modification. Genetic modification may comprise RNA orDNA transfection using any number of techniques known in the art, forexample electroporation (using e.g., the Gene Pulser II, BioRad,Richmond, Calif.), various cationic lipids, (LIPOFECTAMINE™, LifeTechnologies, Carlsbad, Calif.), or other techniques such as calciumphosphate transfection as described in Current Protocols in MolecularBiology, John Wiley & Sons, New York. N.Y. For example, 5-50 μg of RNAor DNA in 500 μl of Opti-MEM can be mixed with a cationic lipid at aconcentration of 10 to 100 μg, and incubated at room temperature for 20to 30 minutes. Other suitable lipids include LIPOFECTIN™,LIPOFECTAMINE™. The resulting nucleic acid-lipid complex is then addedto 1-3×10⁶ cells, preferably 2×10⁶, antigen-presenting cells in a totalvolume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37°C. for 2 to 4 hours. The APC may also be transduced using viraltransduction methodologies as described below.

[0089] In another embodiment of the present invention, APC are loadedwith antigen attached to, coated on, or otherwise immobilized onparticles, such as beads. In the various embodiments, commerciallyavailable beads or other particles, are useful, e.g., MiltenyiParticles, Miltenyi Biotec, Germany; Sepharose beads, Pharmacia FineChemicals, Sweden; DYNABEADS™, Dynal Inc., New York. In certainembodiments, paramagnetic particles or beads are particularly suitable.Such paramagnetic beads or particles are commercially available, forexample, those produced by Dynal AS under the trade name Dynabeads™.Exemplary Dynabeads™ in this regard are M-280, M-450, and M-500. In oneembodiment, whole cells which are live, fixed, irradiated, heat-killedor ohterwise manipulated, are immobilized to ingestable beads, via forexample antibody/ligand specific means or chemical means. Similarly,tumor cell or virus-infected cell lysates, or antigen-preparations canbe attached or otherwise immobilized to the beads (which may beparamagnetic or otherwise selectable). These coated orantigen/cell/lysate-attached beads can be mixed with human or otheranimal peripheral blood preparations (or other compositions containingsome percentage of antigen-presenting cells (particularly those capableof ingesting particles and then processing and presenting antigensassociated with the particles). Phagocytic cells will ingest thebeads/particles, process antigens associated with the particles, andpresent them to T cells in the cell mix. As noted elsewhere herein, onlyT cells with specificity for the variety of presented antigens willinteract in a positive manner with the APC. APC containing paramagneticor otherwise selectable beads can then be isolated carrying with themantigen-specific T cells.

[0090] In one particular embodiment, the particles of the presentinvention comprise a cell surface, such as described in U.S. patentapplication Ser. No. 10/336,224, PCT/US03/00339. In this regard, antigencan be attached to the cells via antibody/ligand specific means asdescribed herein or through genetic modification. Any number oftransfection, transformation, and transduction protocols known to thosein the art may be used, for example those outlined in Current Protocolsin Molecular Biology, John Wiley & Sons, New York. N.Y., or in numerouskits available commercially (e.g., Invitrogen Life Technologies,Carlsbad, Calif.). Such techniques may result in stable transformants ormay be transient. One suitable transfection technique iselectroporation, which may be performed on a variety of cell types,including mammalian cells, yeast cells and bacteria, using commerciallyavailable equipment. Optimal conditions for electroporation (includingvoltage, resistance and pulse length) are experimentally determined forthe particular host cell type, and general guidelines for optimizingelectroporation may be obtained from manufacturers. Other suitablemethods for transfection will depend upon the type of cell used (e.g.,the lithium acetate method for yeast), and will be apparent to those ofordinary skill in the art. Following transfection, cells may bemaintained in conditions that promote expression of the polynucleotidewithin the cell. Appropriate conditions depend upon the expressionsystem and cell type, and will be apparent to those skilled in the art.

[0091] Antigen may be attached to the particles, such as beads, byantibody/ligand specific means, e.g. through particles, such as beads,conjugated to an antibody or antibodies. Suitable antibody/ligand pairsmay include, but are not limited to anti-MART-1 antibody/MART-1 antigen,anti-WT-1 antibody/WT-1, anti-PR1 antibody /PR1, anti-PR3 antibody/PR3,anti-tyrosinase antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1antigen, anti-MUC-1 antibody/MUC-1 antigen, anti-α-fetoproteinantibody/α-fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIVgp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA,anti-CMV pp65/CMV pp65, anti-hepatitis C antibody/hepatitis C proteins,anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-human Ig heavyand lignt chains/Ig from cancer patient, such as myeloma or CLL patient.Other protein:protein binding interactions may be suitable for attachingantigen to particles, such as beads, for example, receptor/ligandinteractions may be utilized. In certain embodiments, theantigen/protein is attached to the particles, such as beads by chemicalmeans, e.g. antigen/protein can be bound through non-covalentassociation of the antigen and bead, simply by incubating/contacting thetwo together for a time and under conditions sufficient for associationto occur. In yet further embodiments, antigen may be attached to theparticles, such as beads by a biotin/avidin or streptavidin interaction.In certain embodiments, hydrophobic “naked” beads withp-toluenesulphonyl (tosyl) reactive groups are used. Proteins areadsorbed hydrophobically on initial coupling with covalent binding ofprimary amine groups (NH₂) and sulphydryl groups (SH) occurringovernight. Coupling reactions can be performed at neutral pH howeverhigh pH and incubation at 37° C. can promote covalent binding.

[0092] In certain aspects, T cells isolated from a tissue source areexposed to antigen-loaded APC described herein for a time sufficient forT cells specific for a given antigen to be activated, for example asdescribed in U.S. Pat. No. 5,827,642, or as described in Riddell, etal., 1990, J. Immunol. Methods, 128:189-201. In one embodiment, T cellsare exposed to antigen-loaded APC for a period of between about severalhours to about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or about 20 days.

[0093] In one embodiment, the T cells are exposed to antigen, orantigen-loaded APC as described herein in vivo. In this regard, antigenor antigen-loaded APC may be administered to an individual in order tostimulate and activate the T cells in vivo. The T cells may then beexpanded either in vivo or ex vivo using the methods as describedherein, such as with anti-CD3/anti-CD28 beads. The quantity andfrequency of administration will be determined by such factors as thecondition of the individual, and the type and severity of disease,although appropriate dosages may be determined by clinical trials. Incertain embodiments the T cells are exposed to antigen in vivo in anindividual prior to onset of a disease or prior to treatment with otherknown therapies. In this regard, the antigen-specific T cells aregenerated and then isolated and expanded and preserved for later use.

[0094] In one embodiment of the present invention, isolation ofantigen-specific T cells in direct contact with APC loaded with antigenimmobilized on particles, such as beads, is performed by magneticisolation of cells which have attached to or engulfed paramagneticparticles. Such separation can be performed using standard methodsavailable in the art. For example, any magnetic separation methodologymay be used including a variety of which are commercially available,(e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®), MACS,Miltenyi Biotec, Germany). In this regard, only T cells with specificityfor the variety of presented antigens will optimally interact in apositive manner with the APC. APC containing paramagnetic (or otherwiseselectable) beads can then be isolated (via magnet or otherwise)carrying with them antigen-specific T cells. These antigen-specific Tcells can then be activated/expanded by a variety of means, such as viaXCELLERATE™ technologies as described herein and U.S. patent applicationSer. Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230;PCT/US01/06139; and PCT/US02/28161.

[0095] In another embodiment of the invention, antigen-specific T cellsare isolated by positive selection. Such isolation can be carried out onT cells freshly isolated from a subject or on T cells that have beenexposed to antigen or antigen-loaded APC as described herein. Numerousimmunoselection methods known to skilled artisans may be used. Suchtechniques are described, for example, in Current Protocols inImmunology, John Wiley & Sons, New York. N.Y. Markers that may be usefulfor the positive selection of antigen-specific cells include, but arenot limited to, CD25, CD54, CD69, CD38, CD45RO, CD49d, CD40L, CD137,CD62L, and CD134. In one embodiment, fluorescence activated cell sortingmay also be used to isolate desired antigen-specific T cells. In anadditional embodiment, antigen-specific T cells may be isolated usingpeptide-MHC tetramers, see for example Altman, et al., Science 1998 Jun.19; 280(5371):1821.

[0096] In a further embodiment of the invention, antigen-specific Tcells may be genetically modified. Genetic modification may comprise RNAor DNA transfection using any number of techniques known in the art, forexample electroporation (using e.g., the Gene Pulser II, BioRad,Richmond, Calif.), various cationic lipids, (LIPOFECTAMINE™, LifeTechnologies, Carlsbad, Calif.), or other techniques such as calciumphosphate transfection as described in Current Protocols in MolecularBiology, John Wiley & Sons, New York. N.Y. For example, 5-50 μg of RNAor DNA in 500 μl of Opti-MEM can be mixed with a cationic lipid at aconcentration of 10 to 100 μg, and incubated at room temperature for 20to 30 minutes. Other suitable lipids include LIPOFECTIN™,LIPOFECTAMINE™. The resulting nucleic acid-lipid complex is then addedto 1-3×10⁶ cells, preferably 2×10⁶, antigen-presenting cells in a totalvolume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37°C. for 2 to 4 hours. The APC may also be transduced using viraltransduction methodologies as described below.

[0097] The antigen-specific T cells of the present invention mayalternatively be genetically modified using retroviral transductiontechnologies. In one aspect of the invention, the retroviral vector maybe an amphotropic retroviral vector, preferably a vector characterizedin that it has a long terminal repeat sequence (LTR), e.g., a retroviralvector derived from the Moloney murine leukemia virus (MoMLV),myeloproliferative sarcoma virus (MPSV), murine embryonic stem cellvirus (MESV), murine stem cell virus (MSCV), spleen focus forming virus(SFFV), or adeno-associated virus (AAV). Most retroviral vectors arederived from murine retroviruses. Retroviruses adaptable for use inaccordance with the present invention can, however, be derived from anyavian or mammalian cell source. These retroviruses are preferablyamphotropic, meaning that they are capable of infecting host cells ofseveral species, including humans. In one embodiment, the gene to beexpressed replaces the retroviral gag, pol and/or env sequences. Anumber of illustrative retroviral systems have been described (e.g.,U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

[0098] In one aspect of the present invention, genetically modifiedantigen-specific T cells can be isolated by any one of numerousimmunoselection methods known to skilled artisans using antibodies orother receptors/ligands specific for the protein or proteins expressedfrom the transgene. Such techniques are known in the art, for example,in Current Protocols in Immunology, John Wiley & Sons, New York. N.Y.

[0099] In one particular embodiment, the antigen-specific T cells may begenetically modified to express a suicide gene, e.g. the herpes simplexvirus thymidine kinase (HSV-TK) as described in Bonini, et al., 1997Science, 276(5319):1719-24, and/or other surface markers (e.g.,truncated nerve growth factor (dNGFR)) for in vivo tracking and/orcontrol of infused antigen-specific T cells. In a further embodiment,the antigen-specific T cells may be genetically modified to express aprotein for targeting the T cells to a particular tissue of interest.

[0100] Those of ordinary skill in the art will readily appreciate thatthe cell separation and culture methodologies described herein, may becarried out in a variety of environments (i.e., containers). Examplesinclude various bags (e.g., Lifecell culture bags), flasks, rollerbottles, bioreactors, (e.g., CellCube (Corning Science Products) orCELL-PHARM, (CD-Medical, Inc. of Hialeah, Fla.)), petri dishes andmulti-well containing plates made for use in tissue culture, or anycontainer capable of holding cells, preferably in a sterile environment.In one embodiment of the present invention a bioreactor is also useful.For example, several manufacturers currently manufacture devices thatcan be used to grow cells and be used in combination with the methods ofthe present invention. See for example, Celdyne Corp., Houston, Tex.;Unisyn Technologies, Hopkinton, Mass.; Synthecon, Inc. Houston, Tex.;Aastrom Biosciences, Inc. Ann Arbor, Mich.; Wave Biotech LLC,Bedminster, N.J. Further, patents covering such bioreactors include U.S.Pat. Nos. 6,096,532; 5,985,653; 5,888,807; 5,190,878.

[0101] Suitable complete growth media for the culture of the APC andantigen-specific T cells of the present invention include for exampleRPMI 1640, DMEM, MEM, α-MEM, AIM-V, HAMS F-12, X-Vivo 15, or X-Vivo 20.In further embodiments, the media can comprise a cytokine, such as IL-2,IFN-γ, IL-4, GM-CSF, IL-10, IL-12, TGFβ, and TNF-α, or a vitamin. Infurther embodiments, the medium comprises surfactant, an antibody,plasmanate or a reducing agent (e.g. N-acetyl-cysteine,2-mercaptoethanol). The growth medium for the cells at each step of themethod of the invention should allow for the survival of the APC and/orthe antigen-specific T cells. Any growth medium typically used toculture cells may be used according to the method of the inventionprovided the medium is supplemented with the appropriate cytokines,serum, antibiotics, vitamins, amino acids or other necessary additives.According to the present invention, the cytokines may be, but are notlimited to, granulocyte-macrophage colony-stimulating factor (GM-CSF)and interleukin 4 (IL-4), or IL-13. Other exemplary cytokines and growthfactors that may be added to the growth medium include but are notlimited to interleukin 1α (IL-1α) and β (IL-1β), IL-2, tumor necrosisfactor alpha (TNF-α), interleukin 3 (IL-3), monocyte colony stimulatingfactor (M-CSF), granulocyte colony-stimulating factor (G-CSF), stem cellfactor (SCF), interleukin 6 (IL-6), interleukin 15 (IL-15), andFlt3-ligand. Preferred media include RPMI 1640, AIM-V, DMEM, MEM, α-MEM,F-12, X-Vivo 15, and X-Vivo 20, with added amino acids and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and an amount of cytokine(s)sufficient to support the expansion of the antigen-specific T cells. Inone aspect, the preferred media comprises 1 liter of X-Vivo 15,BioWhittaker; with 50 ml heat inactivated pooled human serum, 20 ml 1 MHepes, 10 ml 200 mM L-glutamine with or without about 100,000 I.U. IL-2.In one aspect, media may include lipids and/or sources of protein. RPMI1640 supplemented with 1-5% human AB serum preferred. Mixtures ofcytokines may also be used. Cells may also be adapted to grow in othersera, such as fetal calf (bovine) serum (FCS/FBS), at otherconcentrations of serum, or in serum-free media. For example, serum-freemedium supplemented with hormones is also suitable for culturing the APCprecursors. Media may, but does not necessarily, contain antibiotics tominimize growth of bacteria in the cultures. Penicillin, streptomycin orgentamicin or combinations containing them are preferred. The medium, ora portion of the medium, in which the cells are cultured should beperiodically replenished to provide fresh nutrients including GM-CSF,IL-4, IL-13, IL-15 and/or other cytokines.

[0102] Expansion of Antigen-Specific T Cells

[0103] Expansion of the antigen-specific T cells of the presentinvention is carried out by cell surface moiety ligation thatre-stimulates the antigen-specific T cells to proliferate. In oneembodiment of the present invention, the antigen-specific T cells arefirst isolated by methods described herein following exposure to antigenloaded APC. In another embodiment of the present invention, theantigen-specific T cells are expanded directly from the culture withantigen-loaded APC present without an isolation step.

[0104] In one particular embodiment, antigen-specific T cells areactivated and expanded using XCELLERATE™ processes as described hereinand in U.S. patent application Ser. Nos. 10/350,305; 10/187,467;10/133,236; 09/960,264; 09/794,230, with no addition of antigen orantigen-coated particles. In this regard, as noted further herein,antigen-specific T cells that have been previously stimulated oractivated in vivo (e.g. memory T cells) are expanded by an agentproviding a primary activation signal such as an anti-CD3 antibody andan agent providing a co-stimulatory signal, such as an anti-CD28antibody, with both agents co-immobilized to the same surface, such as aparamagnetic bead. As further described herein, see in particular theExamples below, varying the bead:cell ratios during this expansionphase, in particular using low bead:cell ratios, favors expansion ofantigen-specific T cells. For example, bead to cell ratios of 1:200,1:150, 1:125, 1:110, 1:100, 1:75, 1:50, 1:25, 1:20, 1:15, 1:10, 1:5 or1:2.5 are used to expand antigen-specific T cells. A particularadvantage of this aspect of the present invention is that it is notnecessary to add antigen.

[0105] Generally, expansion is carried out by re-stimulating apopulation of antigen-specific T cells and simultaneously stimulating anaccessory molecule on the surface of the antigen-specific T cells with aligand which binds the accessory molecule, as described for example, inU.S. patent application Ser. Nos. 10/350,305, 10/187,467, 10/133,236,09/960,264, 09/794,230, 08/253,694, 08/403,253, 08/435,816, 08/592,711,09/183,055, 09/350,202, and 09/252,150, and U.S. Pat. Nos. 5,858,358;6,352,694; and 5,883,223.

[0106] Generally, re-stimulation may be accomplished by cell surfacemoiety ligation, such as through the T cell receptor (TCR)/CD3 complexor the CD2 surface protein. A number of anti-human CD3 monoclonalantibodies are commercially available, exemplary are, clone BC3 (XR-CD3;Fred Hutchinson Cancer Research Center, Seattle, Wash.), OKT3, preparedfrom hybridoma cells obtained from the American Type Culture Collection,and monoclonal antibody G19-4. Similarly, stimulatory forms of anti-CD2antibodies are known and available. Stimulation through CD2 withanti-CD2 antibodies is typically accomplished using a combination of atleast two different anti-CD2 antibodies. Stimulatory combinations ofanti-CD2 antibodies that have been described include the following: theT11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer etal., Cell 36:897-906, 1984), and the 9.6 antibody (which recognizes thesame epitope as T11.1) in combination with the 9-1 antibody (Yang etal., J. Immunol. 137:1097-1100, 1986). Other antibodies that bind to thesame epitopes as any of the above-described antibodies can also be used.Additional antibodies, or combinations of antibodies, can be preparedand identified by standard techniques. Re-stimulation may also beachieved through contact with antigen, peptide, protein, peptide-MHCtetramers (see Altman, et al Science 1996 Oct. 4; 274(5284):94-6),superantigens (e.g., Staphylococcus enterotoxin A (SEA), Staphylococcusenterotoxin B (SEB), Toxic Shock Syndrome Toxin 1 (TSST-1)), endotoxin,or through a variety of mitogens, including but not limited to,phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin,lipopolysaccharide (LPS), T cell mitogen, and IL-2.

[0107] The antigen-specific cell population may be stimulated orrestimulated as described herein, such as by contact with an anti-CD3antibody or an anti-CD2 antibody immobilized on a surface, or by contactwith a protein kinase C activator (e.g., bryostatin) in conjunction witha calcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T-cells, a ligand that binds the accessory molecule isused. For example, a population of CD4⁺cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T-cells. Similarly, tostimulate proliferation of CD8⁺T-cells, an anti-CD3 antibody and theanti-CD28 antibody B-T3, XR-CD28 (Diaclone, Besançon, France) can beused as can other methods commonly known in the art (Berg et al.,Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

[0108] To further re-stimulate a population of antigen-specific T cells,a co-stimulatory or accessory molecule on the surface of the T cells,such as CD28, is stimulated with a ligand that binds the accessorymolecule. Accordingly, one of ordinary skill in the art will recognizethat any agent, including an anti-CD28 antibody or fragment thereofcapable of cross-linking the CD28 molecule, or a natural ligand for CD28can be used to stimulate T cells. Exemplary anti-CD28 antibodies orfragments thereof useful in the context of the present invention includemonoclonal antibody 9.3 (IgG2_(a)) (Bristol-Myers Squibb, Princeton,N.J.), monoclonal antibody KOLT-2 (IgG1), 15E8 (IgG1), 248.23.2 (IgM),clone B-T3 (XR-CD28; Diaclone, Besançon, France) and EX5.3D10 (IgG2_(a))(ATCC HB11373). Exemplary natural ligands include the B7 family ofproteins, such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al., J.Immunol. 137:3260-3267, 1987; Freeman et al., J. Immunol. 143:2714-2722,1989; Freeman et al., J. Exp. Med. 174:625-631, 1991; Freeman et al.,Science 262:909-911, 1993; Azuma et al., Nature 366:76-79, 1993; Freemanet al., J. Exp. Med. 178:2185-2192, 1993).

[0109] In a further embodiment of the invention, activation of a T-cellpopulation may be enhanced by co-stimulation of other T-cell integralmembrane proteins. For example, binding of the T-cell integrin LFA-1 toits natural ligand, ICAM-1, may enhance activation of cells. Anothercell surface molecule that may act as a co-stimulator for T-cells isVCAM-1 (CD106) that binds very-late-antigen-4 (VLA-4) on T-cells.Ligation of 4-1BB (CD137), a co-stimulatory receptor expressed onactivated T cells, and/or NKG2D may also be useful in the context of thepresent invention to amplify T-cell mediated immunity. It should benoted that more than one costimulatory molecule as described herein maybe stimulated at a time, and in any combination, such that desiredexpansion of the T cells occurs.

[0110] In addition, binding homologues of a natural ligand, whethernative or synthesized by chemical or recombinant techniques, can also beused in accordance with the present invention. Other agents may includenatural and synthetic ligands. Agents may include, but are not limitedto, other antibodies or fragments thereof, a peptide, polypeptide,growth factor, cytokine, chemokine, glycopeptide, soluble receptor,steroid, hormone, mitogen, such as PHA, or other superantigens.

[0111] The primary stimulatory signal and the co-stimulatory signal forthe T-cell may be provided by different protocols. For example, theagents providing each signal may be in solution or coupled to a surface.When coupled to a surface, the agents may be coupled to the same surface(i.e., in “cis” formation) or to separate surfaces (i.e., in “trans”formation). Alternatively, one agent may be coupled to a surface and theother agent in solution. In one embodiment, the agent providing theco-stimulatory signal is bound to a cell surface and the agent providingthe primary activation signal is in solution or coupled to a surface. Incertain embodiments, both agents can be in solution. In anotherembodiment, the agents may be in soluble form, and then cross-linked toa surface, such as a cell expressing Fc receptors or an antibody orother binding agent which will bind to the agents. In a preferredembodiment, the two agents are immobilized on beads, either on the samebead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody and the agent providing the co-stimulatory signal isan anti-CD28 antibody; and both agents are co-immobilized to the samesurface, such as a bead, in equivalent molecular amounts. In oneembodiment, a 1:1 ratio of each antibody bound to the beads for CD4⁺T-cell expansion and T-cell growth is used.

[0112] One aspect of the present invention stems from the surprisingfinding that using lower ratios of anti-CD3:anti-CD28 antibodies boundto the beads results in improved expansion of T cells, includingantigen-specific T cells. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 0.5 to about 3 fold is observed as compared to theexpansion observed using a ratio of 1:1. In one embodiment, the ratio ofCD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and allinteger values there between. In one aspect of the present invention,more anti-CD28 antibody is bound to the particles than anti-CD3antibody, i.e. the ratio of CD3:CD28 is less than one. In certainembodiments of the invention, the ratio of anti CD28 antibody to antiCD3 antibody bound to the beads is greater than 2:1. In one particularembodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used.In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beadsis used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio ofantibody bound to beads is used. In one preferred embodiment, a 1:10CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used.In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to thebeads is used.

[0113] Ratios of particles to cells from 1:500 to 500:1 and any integervalues in between may be used to stimulate T-cells. As those of ordinaryskill in the art can readily appreciate, the ratio of particle to cellsmay dependant on particle size relative to the target cell. For example,small sized beads could only bind a few cells, while larger beads couldbind many. In certain embodiments the ratio of particles to cells rangesfrom 1:100 to 100:1 and any integer values in-between and in furtherembodiments the ratio comprises 1:9 to 9:1 and any integer values inbetween, can also be used to stimulate T-cells. The ratio of anti-CD3-and anti-CD28-coupled particles to T-cells that result in T-cellstimulation and expansion can vary as noted above, however in certainembodiments the ratio may be 1:150 or lower. Certain preferred ratiosinclude 1:150, 1:100, 1:75, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15, 1:10,1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, and 20:1 with one preferred ratio being1:1 particles per T-cell. In one embodiment, a ratio of particles tocells of 1:1 or less is used. In one particular embodiment, a preferredparticle:cell ratio is 1:2.5 or 1:5. In further embodiments, the ratioof particles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:5, 1:2.5, 1:1 to 10:1 on the first day and additional particles areadded to the cells every day or every other day thereafter for up to 10days, at final ratios of from 1:1, 1:5, 1:20, 1:25, 1:50, or 1:100(based on cell counts on the day of addition). In one particularembodiment, the ratio of particles to cells is 1:2.5, 1:5, or 1:1 on thefirst day of stimulation and adjusted to 1:5 on the third and fifth daysof stimulation. In a further embodiment, the ratio of particles to cellsis 1:2.5, 1:5, or 1:1 on the first day of stimulation and adjusted to1:10, 1:20, 1:25, 1:50, or 1:100 at day 5, 7, or 9. In anotherembodiment, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In another embodiment, the ratio of particles to cellsis 2:1 on the first day of stimulation and adjusted to 1:10 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:10 on the third and fifth days of stimulation. One ofskill in the art will appreciate that a variety of other ratios may besuitable for use in the present invention.

[0114] One aspect of the present invention stems from the surprisingfinding that using different bead:cell ratios can lead to differentoutcomes with respect to expansion of antigen-specific T cells. Inparticular, bead:cell ratios can be varied to selectively expand ordelete antigen-specific (memory) T cells. In one embodiment, theparticular bead:cell ratio used selectively deletes antigen-specific Tcells. In a further embodiment, the particular bead:cell ratio usedselectively expands antigen-specific T cells. For example, bead to cellratios of 1:100, 1:50, 1:25, 1:5 or 1:2.5 and the like are used toexpand antigen-specific T cells. Low bead:cell ratio can help preserveand promote expansion of memory (antigen-specific) T cells.Additionally, when additional beads are added at very low ratios tocells (1:10, 1:25, 1:50, 1:100) at various days of culture (e.g.sequential addition at day 5, 7, or 9), one can enhance and even promotepreferential expansion of the memory cells. With either 1:5 or 1:2.5bead:cell ratio as initial simulus, addition of 1:10, 1:25, and to someextent 1:50 and 1:100 bead:cell raio at days 5 and 7 appear to preserveand enhance further expansion of memory cells that would otherwise notoccur with a single stimulation at day 0 (see specifically Examplesdescribed herein). Therefore, the compositions and methods describedherein can be used to expand specific populations of T cells, or todelete specific populations of T cells, for use in any variety ofimmunotherapeutic settings described herein.

[0115] It should be noted that the particle:cell ratios described hereincan be used in any combination with the various ratios of antibodiesbound on the beads. For example, beads containing about 1:5 to 1:10ratio of anti-CD3/anti-CD28 antibodies bound thereto can be used at aratio of about 1:5 to 1:10 particles:cell. Or, beads containing a 1:1ratio of anti-CD3/anti-CD28 antibodies bound thereto can be used at aratio of about 1:5 particles:cell, etc. Thus, the ratio ofanti-CD3:anti-CD28 antibody bound to the beads ranges from 100:1 to1:100 and all integer values there between and such beads can be used ata ratio of particle:cell of anywhere from about 1:500 to 500:1 and anyinteger values in between, in any combination.

[0116] Using certain methodologies it may be advantageous to maintainlong-term stimulation of a population of T-cells following the initialactivation and stimulation, by separating the T-cells from the stimulusafter a period of about 12 to about 14 days. The rate of T-cellproliferation is monitored periodically (e.g., daily) by, for example,examining the size or measuring the volume of the T-cells, such as witha Coulter Counter. In this regard, a resting T-cell has a mean diameterof about 6.8 microns, and upon initial activation and stimulation, inthe presence of the stimulating ligand, the T-cell mean diameter willincrease to over 12 microns by day 4 and begin to decrease by about day6. When the mean T-cell diameter decreases to approximately 8 microns,the T-cells may be reactivated and re-stimulated to induce furtherproliferation of the T-cells. Alternatively, the rate of T-cellproliferation and time for T-cell re-stimulation can be monitored byassaying for the presence of cell surface molecules, such as, CD154,CD54, CD25, CD137, CD134, which are induced on activated T-cells.

[0117] In one embodiment, T-cell stimulation is performed with anti-CD3and anti-CD28 antibodies co-immobilized on beads (3×28 beads), for aperiod of time sufficient for the cells to return to a quiescent state(low or no proliferation) (approximately 8-14 days after initialstimulation). The stimulation signal is then removed from the cells andthe cells are washed and infused back into the patient. The cells at theend of the stimulation phase are rendered “super-inducible” by themethods of the present invention, as demonstrated by their ability torespond to antigens and the ability of these cells to demonstrate amemory-like phenotype, as is evidence by the examples. Accordingly, uponre-stimulation either exogenously or by an antigen in vivo afterinfusion, the activated T-cells demonstrate a robust responsecharacterized by unique phenotypic properties, such as sustained CD154expression and increased cytokine production.

[0118] In further embodiments of the present invention, the cells, suchas T-cells, are combined with agent-coated beads, the beads and thecells are subsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, resulting in cell surface moiety ligation, thereby inducingcell stimulation.

[0119] In another embodiment, the time of exposure to stimulatory agentssuch as anti-CD3/anti-CD28 (i.e., CD3xCD28)-coated particles, such asbeads, may be modified or tailored to obtain a desired T-cell phenotype.One may desire a greater population of helper T-cells (T_(H)), typicallyCD4⁺ as opposed to CD8⁺ cytotoxic or suppressor T-cells (T_(C)), becausean expansion of T_(H) cells could induce desired effector function(e.g., anti-tumor, anti-viral, anti-bacterial, and the like). CD4⁺T-cells, express important immune-regulatory molecules, such as GM-CSF,CD40L, and IL-2, for example. Where CD4-mediated help is preferred, amethod, such as that described herein, which preserves or enhances theCD4:CD8 ratio could be of significant benefit. In one aspect of thepresent invention, it may be beneficial to increase the number ofinfused cells expressing GM-CSF, or IL-2, all of which are expressedpredominantly by CD4⁺ T-cells. Alternatively, in situations whereCD4-help is needed less and increased numbers of CD8⁺T-cells aredesirous, the T cell activation approaches described herein can also beutilized, by for example, pre-selecting for CD8⁺ cells prior tostimulation and/or culture. Such situations may exist where increasedlevels of IFN-γ is preferred. Further, in other applications, it may bedesirable to utilize a population of T_(H)1-type cells versusT_(H)2-type cells (or vice versa), or supernatants therefrom. Likewise,it may be desirable in certain applications to utilize a population ofregulatory T cells (e.g., Autoimmun Rev. 2002 August;1(4):190-7; CurrOpin Immunol. 2002 December; 14(6):771-8).

[0120] To effectuate isolation of different antigen-specific T-cellpopulations, times of cell surface moiety ligation that inducesre-stimulation (activation) may be varied or pulsed. For exampleexpansion times may be varied to obtain the specific phenotype ofinterest and/or different types of stimulatory agents may be used (e.g.,antibodies or fragments thereof, a peptide, polypeptide, MHC/peptidetetramer, growth factor, cytokine, chemokine, glycopeptide, solublereceptor, steroid, hormone, mitogen, such as PHA, or othersuperantigens). The expression of a variety of phenotypic markers changeover time; therefore, a particular time point or stimulatory agent maybe chosen to obtain a specific population of T-cells. Accordingly,depending on the cell type to be stimulated, the stimulation and/orexpansion time may be four weeks or less, 2 weeks or less, 10 days orless, or 8 days or less (four weeks or less includes all time rangesfrom 4 weeks down to 1 day (24 hours)). In some embodiments, stimulationand expansion may be carried out for 6 days or less, 4 days or less, 2days or less, and in other embodiments for as little as 24 or lesshours, and preferably 4-6 hours or less (these ranges include anyinteger values in between). When stimulation of T-cells is carried outfor shorter periods of time, the population of T-cells may not increasein number as dramatically, but the population will provide robust andhealthy activated antigen-specific T-cells that can continue toproliferate in vivo and more closely resemble the natural effectorT-cell pool.

[0121] In one embodiment of the present invention, the mixture may becultured for several hours (about 3 hours) to about 14 days or anyhourly integer value in between. In another embodiment, the mixture maybe cultured for 21 days. In one embodiment of the invention the beadsand the T-cells are cultured together for about eight days. In anotherembodiment, the beads and T-cells are cultured together for 2-3 days.Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T-cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (BioWhittaker)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,GM-CSF, IL-10, IL-12, TGFβ, and TNF-α. or any other additives for thegrowth of cells known to the skilled artisan. Other additives for thegrowth of cells include, but are not limited to, surfactant, plasmanate,and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15,and X-Vivo 20, with added amino acids and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T-cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO₂).

[0122] In certain embodiments, it may be desirable to add some number offeeder cells to augment activation and/or expansion of antigen-specificcells. Feeder cells can encompass a variety of cell types, including,irradiated peripheral blood lymphocytes (autologous or allogeneic) aloneor in combination with EBV-transformed B cell lines (autologous orallogeneic), immortalized or non-immortalized cell lines of themyelomoncytic lineage, such as macrophges, dentritic cells, red bloodcells, B-cells, tumor cell lines such as U937, Jurkat, Daudi, MOLT-4,HUT, CEM, Colo 205, HTB-13, and HTB-70. Feeder cells need not be ofhuman origin as long as they provide feeder function, e.g. the abilityto facilitate the survival and growth of primary T cells and therderived antigen-specific clones.

[0123] Pharmaceutical Compositions

[0124] An additional aspect of the present invention provides apopulation or composition of antigen-specific T cells. The presentinvention further provides a pharmaceutical composition comprisingantigen-specific T cells and a pharmaceutically acceptable carrier.Compositions of the present invention may be administered either alone,or as a pharmaceutical composition in combination with diluents and/orwith other components such as IL-2 or other cytokines or cellpopulations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such asethylenediaminetetraacetic acid (EDTA) or glutathione; adjuvants (e.g.,aluminum hydroxide); and preservatives. Compositions of the presentinvention are, in certain aspects, formulated for intravenousadministration.

[0125] A related embodiment of the present invention further provides apharmaceutical composition comprising the antigen-specific T cells, anda pharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier should be sterilized by techniques known to those skilled in theart.

[0126] Pharmaceutical compositions of the present invention may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

[0127] The present invention also provides methods for preventing,inhibiting, or reducing the presence of a cancer or malignant cells inan animal, which comprise administering to an animal an anti-cancereffective amount of the subject antigen-specific T cells.

[0128] The cancers contemplated by the present invention, against whichthe immune response is induced, or which is to be prevented, inhibited,or reduced in presence, may include but are not limited to melanoma,non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectalcancer, kidney cancer, renal cell carcinoma, pancreatic cancer,esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervicalcancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), low-grade lymphoma, and otherneoplasms known in the art.

[0129] Alternatively, compositions as described herein can be used toinduce or enhance responsiveness to pathogenic organisms, such asviruses, (e.g., single stranded RNA viruses, single stranded DNAviruses, human immunodeficiency virus (HIV), hepatitis A, B, and Cvirus, herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barrvirus (EBV), Human Papilloma Virus (HPV)), parasites (e.g., protozoanand metazoan pathogens such as Plasmodia species, Leishmania species,Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria,Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candidaspecies, Aspergillus species) and Pneumocystis carinii.

[0130] In certain embodiments, the methods of the present invention canbe used in conjunction with the generation of T regulatory cells forspecific immunosuppression in the case of inflammatory disease,autoimmunity, and foreign graft acceptance. Regulatory T cells can begenerated and expanded using the methods of the present invention. Theregulatory T cells can be antigen-specific and/or polyclonal. RegulatoryT cells can be generated using art-recognized techniques as describedfor example, in Woo, et al., J. Immunol. 2002 May 1;168(9):4272-6;Shevach, E. M., Annu. Rev. Immunol. 2000, 18:423; Stephens, et al., Eur.J. Immunol. 2001, 31:1247; Salomon, et al, Immunity 2000, 12:431; andSakaguchi, et al., Immunol. Rev. 2001, 182:18. Accordingly, T cells ofthe present invention can be used for the treatment of autoimmunediseases such as, but not limited to, rheumatoid arthritis, multiplesclerosis, insulin dependent diabetes, Addison's disease, celiacdisease, chronic fatigue syndrome, inflammatory bowel disease,ulcerativecolitis, Crohn's disease, Fibromyalgia, systemic lupuserythematosus, psoriasis, Sjogren's syndrome, hyperthyroidism/Gravesdisease, hypothyroidism/Hashimoto's disease, Insulin-dependent diabetes(type 1), Myasthenia Gravis, endometriosis, scleroderma, perniciousanemia, Goodpasture syndrome, Wegener's disease, glomerulonephritis,aplastic anemia, paroxysmal nocturnal hemoglobinuria, myelodysplasticsyndrome, idiopathic thrombocytopenic purpura, autoimmune hemolyticanemia, Evan's syndrome, Factor VIII inhibitor syndrome, systemicvasculitis, dermatomyositis, polymyositis and rheumatic fever.

[0131] The immune response induced in the animal by administering thesubject compositions of the present invention may include cellularimmune responses mediated by cytotoxic T cells, capable of killing tumorand infected cells, and helper T cell responses. Humoral immuneresponses, mediated primarily by helper T cells capable of activating Bcells thus leading to antibody production, may also be induced. Avariety of techniques may be used for analyzing the type of immuneresponses induced by the compositions of the present invention, whichare well described in the art; e.g., Coligan et al. Current Protocols inImmunology, John Wiley & Sons Inc. (1994).

[0132] When “an immunologically effective amount”, “an anti-tumoreffective amount”, “an tumor-inhibiting effective amount”, or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient. It can generally be stated that apharmaceutical composition comprising the subject antigen-specific Tcells, may be administered at a dosage of 10⁴ to 10⁷ APC/kg body weight,preferably 10⁵ to 10⁶ APC/kg body weight, including all integer valueswithin those ranges. Antigen-specific T cells compositions may also beadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

[0133] Typically, in adoptive immunotherapy studies, antigen-specific Tcells are administered approximately at 2×10⁹ to 2×10¹¹ cells to thepatient. (See, e.g., U.S. Pat. No. 5,057,423). In some aspects of thepresent invention, particularly in the use of allogeneic or xenogeneiccells, lower numbers of cells, in the range of 10⁶/kilogram (10⁶-10¹¹per patient) may be administered. In certain embodiments, T cells areadministered at 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 2×10⁸, 2×10⁹, 1×10¹⁰,2×10¹⁰, 1×10¹¹, 5×10¹¹, or 1×10¹² cells to the subject. T cellcompositions may be administered multiple times at dosages within theseranges. The antigen-specific T cells may be autologous or heterologousto the patient undergoing therapy. If desired, the treatment may alsoinclude administration of mitogens (e.g., PHA) or lymphokines,cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES,MIP1α, etc.) as described herein to enhance induction of the immuneresponse.

[0134] The administration of the subject pharmaceutical compositions maybe carried out in any convenient manner, including by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions of the present invention may beadministered to a patient subcutaneously, intradermally,intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.In one embodiment, the antigen-specific T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the antigen-specific Tcell compositions of the present invention are preferably administeredby i.v. injection. The compositions of antigen-specific T cells may beinjected directly into a tumor or lymph node.

[0135] In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC Crit.Ref. Biomed. Eng. 14:201; Buchwald et al., 1980; Surgery 88:507; Saudeket al., 1989, N. Engl. J. Med. 321:574). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.;Controlled Drug Bioavailability, Drug Product Design and Performance,1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983;J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howardet al., 1989, J. Neurosurg. 71:105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Medical Applications of Controlled Release, 1984, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).

[0136] The antigen-specific T cell compositions of the present inventionmay also be administered using any number of matrices. Matrices havebeen utilized for a number of years within the context of tissueengineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer,and Chick (eds.)), 1997. The present invention utilizes such matriceswithin the novel context of acting as an artificial lymphoid organ tosupport, maintain, or modulate the immune system, typically throughmodulation of T cells. Accordingly, the present invention can utilizethose matrix compositions and formulations which have demonstratedutility in tissue engineering. Accordingly, the type of matrix that maybe used in the compositions, devices and methods of the invention isvirtually limitless and may include both biological and syntheticmatrices. In one particular example, the compositions and devices setforth by U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069;5,787,900; or 5,626,561 are utilized. Matrices comprise featurescommonly associated with being biocompatible when administered to amammalian host. Matrices may be formed from both natural or syntheticmaterials. The matrices may be non-biodegradable in instances where itis desirable to leave permanent structures or removable structures inthe body of an animal, such as an implant; or biodegradable. Thematrices may take the form of sponges, implants, tubes, telfa pads,fibers, hollow fibers, lyophilized components, gels, powders, porouscompositions, or nanoparticles. In addition, matrices can be designed toallow for sustained release seeded cells or produced cytokine or otheractive agent. In certain embodiments, the matrix of the presentinvention is flexible and elastic, and may be described as a semisolidscaffold that is permeable to substances such as inorganic salts,aqueous fluids and dissolved gaseous agents including oxygen.

[0137] A matrix is used herein as an example of a biocompatiblesubstance. However, the current invention is not limited to matrices andthus, wherever the term matrix or matrices appears these terms should beread to include devices and other substances which allow for cellularretention or cellular traversal, are biocompatible, and are capable ofallowing traversal of macromolecules either directly through thesubstance such that the substance itself is a semi-permeable membrane orused in conjunction with a particular semi-permeable substance.

[0138] In certain embodiments of the present invention, the cells of thepresent invention are administered to a patient in conjunction with(e.g. before, simulataneously or following) any number of relevanttreatment modalities, including but not limited to treatment with agentssuch as antiviral agents, chemotherapy, radiation, immunosuppressiveagents, such as cyclosporin, azathioprine, methotrexate, mycophenolate,and FK506, antibodies, or other immunoablative agents such as CAMPATH,anti-CD3 antibodies, cytoxin, fludaribine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. Thesedrugs inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (supra)). In a furtherembodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g. before,simulataneously or following) T-cell ablative therapy using eitherchemotherapy agents such as, fludarabine, external-beam radiationtherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.In another embodiment, the cell compositions of the present inventionare administered following B-cell ablative therapy such as agents thatreact with CD20, e.g. Rituxan. The dosage of the above treatments to beadministered to a patient will vary with the precise nature of thecondition being treated and the recipient of the treatment. The scalingof dosages for human administration can be performed according toart-accepted practices. The dose for CAMPATH, for example, willgenerally be in the range 1 to about 100 mg for an adult patient,usually administered daily for a period between 1 and 30 days. Thepreferred daily dose is 1 to 10 mg per day although in some instanceslarger doses of up to 40 mg per day may be used (described in U.S. Pat.No. 6,120,766.

[0139] All references referred to within the text are herebyincorporated by reference in their entirety. Moreover, all numericalranges utilized herein explicitly include all integer values within therange and selection of specific numerical values within the range iscontemplated depending on the particular use. Further, the followingexamples are offered by way of illustration, and not by way oflimitation.

EXAMPLES Example 1 CMV ANTIGEN COATED BEADS ACTIVATE AND FACILITATEISOLATION OF ANTIGEN-SPECIFIC T CELLS

[0140] In this experiment, cytomegalovirus (CMV)-coated beads were usedto activate and isolate antigen-specific T cells.

[0141] CMV lysate prepared using standard techniques was mixed at roomtemperature for 1-2 hours with Dynabead M-450 while rotating. Beads werethen washed once, and added to PBMC. Within hours, the beads werephagocytosed in the APC. Within 72 hours, CMVpp65-HLA-A2 tetramersdetected CD25-high (activated) T cell specific for CMV pp65. Magneticselection of the bead-loaded APC with the associated antigen-specific Tcells was carried out at day 5, thereby enriching for CMV-specific Tcells. As shown in FIG. 1, following magnetic separation, CMV-specific Tcells were still tightly associated with bead-loaded APC. It should benoted that magnetic separation can be carried out anywhere from aboutday 1 to about day 10.

Example 2 MEMORY CD8 CMV TETRAMER ⁺ T CELLS EXPANDED EX VIVO UP-REGULATECD25 UPON R E-STIMULATION

[0142] In this example, antigen-coated beads were used to activateCMV-specific CD8⁺T cells ex vivo.

[0143] PBMC from CMV pp65 tetramer-positive and tetramer-negative donorswere stimulated with paramagnetic Dynal M-450 beads coated with CMVlysate. As controls, CMV pp65 tetramer-negative PBMC were cultured withCMV-lysate coated beads (FIG. 2, panel A), CMV pp65 tetramer-positivePBMC were cultured with “naked” beads (no CMV antigen) (FIG. 2, panelB). CMV pp65 tetramer-positive PBMC were cultured with CMV-lysate coatedbeads (FIG. 2, panel C). Following stimulation, activation ofCMV-specific T cells was measured on Day 10 by CMV pp65 HLA-A2 tetramerstain and CD25 expression as an indicator of activation. As shown inFIG. 2, up-regulation of CD25 was observed in memory CD8 CMV tetramer+Tcells expanded ex vivo using antigen-coated beads.

[0144] Antigen-coated beads can be used to activate and stimulateantigen-specific T cells. These antigen-specific T cells can then beenriched as described in Example 1 and elsewhere herein. Theseantigen-specific T cells can be further expanded as described herein andin U.S. patent application Ser. Nos. 10/350,305, 10/187,467, 10/133,236,09/960,264, and 09/794,230. The antigen-specific T cells of the presentinvention can be used in any number of immunotherapeutic settings asdescribed herein.

Example 3 VARYING BEAD:CELL RATIOS CAN SELECTIVELY EXPAND OR DELETEMEMORY CD8 T CELLS

[0145] This example shows that the bead:cell ratio can have a profoundeffect on expansion of different populations of T cells. In particular,a high bead:cell ratio (3:1-10:1, 20:1 and higher) tends to induce deathin antigen-specific T cells while a lower bead:cell ratio (1:1-1:10,1:20, 1:30, 1:40, 1:50 or lower) leads to expansion of antigen-specificT cells. Further, the data described below show that lower bead:cellratios lead to improved cell expansion in polyclonal cell populations aswell. Thus, this example shows that lower bead:cell ratios improveoverall cell expansion.

[0146] Cells were prepared and stimulated using the XCELLERATE I™process essentially as described in U.S. patent application Ser. No.10/187,467 filed Jun. 28, 2002. Briefly, in this process, theXCELLERATED™ T-cells are manufactured from a peripheral bloodmononuclear cell (PBMC) apheresis product. After collection from thepatient at the clinical site, the PBMC apheresis are washed and thenincubated with “uncoated” DYNABEADS® M-450 Epoxy T. During this timephagocytic cells such as monocytes ingest the beads. After theincubation, the cells and beads are processed over a MaxSep MagneticSeparator in order to remove the beads and any monocytic/phagocyticcells that are attached to the beads. Following this monocyte-depletionstep, a volume containing a total of 5×10⁸ CD3⁺ T-cells is taken andset-up with 1.5×10⁹ DYNABEADS® M-450 CD3/CD28 T to initiate theXCELLERATE™ process (approx. 3:1 beads to T-cells). The mixture of cellsand DYNABEADS® M-450 CD3/CD28 T are then incubated at 37° C., 5% CO₂ forapproximately 8 days to generate XCELLERATED T-cells for a firstinfusion. The remaining monocyte-depleted PBMC are cryopreserved until asecond or further cell product expansion (approximately 21 days later)at which time they are thawed, washed and then a volume containing atotal of 5×10⁸ CD3⁺ T-cells is taken and set-up with 1.5×10⁹ DYNABEADS®M-450 CD3/CD28 T to initiate the XCELLERATE Process for a secondinfusion. During the incubation period of ≈8 days at 37° C., 5% CO₂, theCD3⁺T-cells activate and expand. The anti-CD3 mAb used is BC3 (XR-CD3;Fred Hutchinson Cancer Research Center, Seattle, Wash.), and theanti-CD28 mAb (B-T3, XR-CD28) is obtained from Diaclone, Besançon,France.

[0147] For the experiment described below, prior to plating andculturing, the monocyte depleted cells were mixed by rotation for 30minutes with varying amounts of beads as summarized below in Table 1.The beads used in this Example comprised the DYNABEADS® M-450 CD3CD28 Twith a 1:1 CD3:CD28 antibody ratio bound on the beads. TABLE 1 VaryingBead:Cell Ratios can Selectively Expand or Delete Memory CD8 T cellsFold Increase Bead:Cell Ratio Polyclonal T cells CMV Antigen-Specific Tcells 10:1  149 0 5:1 294 0 3:1 346 1.4 1:1 562 20.6 1:5 113 53  1:10 7945.8

[0148] The results summarized in Table 1 and shown graphically in FIG. 3demonstrate that antigen-specific T cells can be selectively deleted byusing high bead:cell ratios and expanded using low bead:cell ratios(similar results were observed with Influenza- and EBV-specific cells).Without being bound by theory, it is thought that the antigen-specific Tcells are sensitized to further stimulation. Thus, the key appears to bethe strength of the T cell activation signal: selective expansion ofmemory T cells (antigen-specific T cells) occurs with “weak” signalswhile selective deletion of memory T cells occurs with “strong” signals.The quantity of the CD3/TCR (and CD28) receptors that bound by ligandsdetermines the signal strength. Thus, stimulation with high bead:cellratios provides a high concentration of stimulating antibody (i.e.,“strong signal”), leading to over-stimulation of antigen-specific Tcells, causing them to die, either by apoptosis or other mechanisms.Using lower bead:cell ratios provides a stimulation signal toantigen-specific T cells that does not over-stimulate, but ratherinduces rapid proliferation of these cells.

[0149] In further experiments, fold increase of antigen-specific (e.g.,CMV tetramer positive cells) was shown to be excellent using a 1:30ratio and also using beads bound with anti-4-1 BB antibody.

[0150] Therefore, in this Example, evidence is provided to support theuse of differing bead:cell ratios depending on the outcome desired. Forexpansion of antigen-specific T cells, a lower bead:cell ratio ispreferable.

Example 4 VARYING BEAD:CELL RATIOS AND SEQUENTIAL ADDITION OF BEADSDURING CULTURE CAN IMPROVE EXPANSION OF MEMORY T CELLS

[0151] This example shows that sequential addition of beads at a lowbead:cell ratio during culture can improve expansion of memory T cells.

[0152] Cells were prepared and stimulated essentially as described inExample 3 with the following modifications: as shown in FIG. 4, panels Aand B, cells were cultured either at a starting static culture with abead:cell ratio of 1:2.5 or 1:5 OR at 1:2.5 or 1:5 starting ratio withadditional beads added at day 5, 7, or 9 at 1:10, 1:25, 1:50 or 1:100ratios as noted. A comparison of total T cell expansion over 15 daysshows an increase in expansion of cells when beads are addedsequentially over culturing time, in cultures with both startingbead:cell ratios of 1:2.5 and 1:5. Comparison of CMV-specific T cellexpansion over 15 days also shows an increase in expansion ofantigen-specific cells when beads are added sequentially during culture(see FIG. 4 panel A and FIG. 4 panel B). The most dramatic increase inexpansion of polyclonal cells and antigen-specific T cells over staticculture was observed in those cultures where beads were added at day 0at a ratio of 1:2.5 beads:cells and sequentially added at a 1:10 ratioat day 5.

[0153] In a related experiment, reduced bead:cell ratio and sequentialaddition was used to examine expansion of T cells from patientsvaccinated with Melanoma gp100(M). As shown in FIG. 5, using a reducedbead:T cell ratio of 1:50 and sequential addition at days 3, 5, 11, 15,and 19, a dramatic increase in expansion was observed in Melanomagp100(M)-specific T cells.

Example 5 ASSESSMENT OF CD4+ T M EMORY (“ANTIGEN-EXPERIENCED”) T CELLSIN THE XCELLERATE EXPANSION PROCESS

[0154] This example describes a model system for assessing CD4 T cellsubsets in the Xcellerate® expansion process.

[0155] Toxic Shock Syndrome Toxin (TSST) is a superantigen thatspecifically stimulates CD4+ T cells expressing TCR Vβ2. PBMC arecomposed of between 1-25% Vβ2 TCR T cells. A CD4⁺ Vβ2 specific cell lineis generated by stimulating PBMC with TSST for 9-14 days until T cellsproliferate out of log phase. These “antigen experienced” Vβ2 T cellsare then mixed back at varying percentages of the total culture (e.g.,1%, 2%) with a Vβ2 depleted naïve PBMC culture and stimulated withCD3/CD28 beads at varying bead:cell ratios as described herein in theXcellerate® process.

[0156] The results showed that the presence of TSST expanded CD4+ VP2TCR T cells does not inhibit total T cell Xcellerate® expansion, withtotal T cell fold increases in the normal range. Further, confirmingother experiments, antigen-specificity was maintained during expansionand antigen experienced Vβ2 TCR T cells expanded well at bead:cellratios of 1:10 and 1:30.

Example 6 T CELL EXPANSION USING VARYING ANTI-CD3:ANTI-CD28 ANTIBODYRATIOS

[0157] T cell expansion was evaluated using varying concentrations ofanti-CD3:anti-CD28 antibody ratios on the 3×28 DYNABEADS® M-450. In theexperiments described herein, the process referred to as XCELLERATE II™was used, as described in U.S. patent application Ser. No. 10/187,467.Briefly, this process is similar to XCELLERATE I™ as described inExample 3 with some modifications in which no separate monocytedepletion step was utilized and in certain processes the cells werefrozen prior to initial contact with beads and further concentration andstimulation were performed. As shown in FIG. 6, surprisingly, about a68-fold expansion after 8 days of culture was observed with ananti-CD3:CD28 ratio of 1:10 antibodies on the beads. A 35-fold expansionof T cells was seen after 8 days of culture with a CD3:CD28 ratio of 1:3on the beads. At a 1:1 ratio, about a 24-fold expansion was seen. Asshown in FIG. 7, similar results were observed with CMVpp65-specificCD8⁺T cells using anti-CD3:anti-CD28 antibody ratios as low as 1:30.

Example 7 T CELL EXPANSION USING THE XCELLERATE PROCESS AND THE WAVEBIOREACTOR

[0158] This example describes the T cells expansion using essentiallythe Xcellerate II process as described in U.S. Patent Application Nos.10/350,305; 10/187,467; 10/133,236; 09/960,264; 09/794,230;PCT/US01/06139; and PCT/US02/28161, followed by seeding cells into theWave Bioreactor.

[0159] Day 0 of the Xcellerate Process—On the first day of theXcellerate process essentially, the required number of cryopreservedCryocte™ containers from were removed from the storage freezer, thawedwashed and filtered.

[0160] Day 0—A volume of cells containing approximately 0.5×10⁹ CD3⁺cells was then mixed with Dynabeads M-450 CD3/CD28 T at a ratio of 3:1Dynabeads M-450 CD3/CD28 T:CD3⁺ T cells and incubated with rotation.After the incubation, the CD3⁺ T cells were magnetically concentratedand simultaneously activated. The CD3⁺ T cells were then resuspended incomplete medium in a Lifecell Cell Culture Bag. The bag containing thecells and beads was then placed in a patient-dedicated incubator (37°C., 5% CO₂).

[0161] On or around Day 3—The CD3⁺ cells were culture-expanded for ≈3days at which point the contents of the single bag are split into 4 newLifecell bags. The 4 bags were then returned to the patient-dedicatedincubator (37° C., 5% CO₂).

[0162] On or around Day 5—The CD3⁺ cells were culture-expanded for ≈2additional days at which point the contents of the culture bags werethen seeded into a 20 L Wave Bioreactor containing a 10 L volume ofmedia. The cells were then cultured at 37° C., 5% CO₂ with the wavemotion at 15 rocks/minute and with perfusion at 1 ml/minute.

[0163] Cell counts were determined each day and compared to cellsstimulated and expanded using the static Xcellerate II process.Expansion was dramatically improved when cells were cultured in The WaveBioreactor. Further, cell densities reached as high as 50×10⁶ cells/mlin The Wave Bioreactor, as compared to a maximum cell density of 5×10⁶observed in the static Xcellerate II process. A total cell count ofabout 800 billion was achieved at day 12 of culture from a starting cellcount of about 0.5×10⁹ cells using The Wave Bioreactor.

[0164] Thus, The Wave Bioreactor provides an unexpected and dramaticimprovement to the expansion process. Furthermore, hitherto unobservedcell densities and final absolute cell yields were achieved using TheWave Bioreactor.

[0165] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for expanding a population of antigen-specific T cellscomprising: contacting a population of cells wherein at least a portionthereof comprises antigen-specific T cells, with a surface, wherein saidsurface has attached thereto a first agent and a second agent, whereinsaid first agent ligates a CD3/TCR complex on said T cells and saidsecond agent ligates an accessory molecule on said T cells, and whereinsaid ligation by said first and second agent of said T cells inducesproliferation of antigen-specific T cells and wherein said surface ispresent at a ratio of surface to T cells of 1:2 or less.
 2. The methodaccording to claim 1 wherein said surface is selected from the groupconsisting of paramagnetic beads, lipids, and cell surfaces.
 3. Themethod according to claim 2 wherein said surface comprises paramagneticbeads.
 4. The method according to claim 3 wherein said beads comprisebeads conjugated to an antibody.
 5. The method of claim 1 wherein saidsurface is present in a ratio of surface to T cells of about 1:2.5. 6.The method of claim 1 wherein said surface is present in a ratio ofsurface to T cells of about 1:5.
 7. The method of claim 1 wherein saidsurface is present in a ratio of surface to T cells of about 1:10. 8.The method of claim 1 wherein said surface is present in a ratio ofsurface to T cells of about 1:25.
 9. The method of claim 1 wherein saidsurface is present in a ratio of surface to T cells of about 1:50. 10.The method of claim 1 wherein said surface is present in a ratio ofsurface to T cells of about 1:100.
 11. A method for generating and/orenriching antigen-specific T cells comprising: (a) exposing a firstpopulation of cells wherein at least a portion thereof comprises antigenpresenting cells to a surface wherein said surface has antigen attachedthereto, such that said surface with antigen attached thereto isingested by said APC; (b) exposing a second population of cells whereinat least a portion thereof comprises T cells to the population of cellsin part (a); thereby generating and/or enriching antigen-specific Tcells.
 12. The method according to claim 11 wherein said APC are indirect contact with said antigen-specific T cells.
 13. The methodaccording to claim 12 wherein said APC in direct contact with saidantigen-specific T cells are isolated by exposing said APC to a magneticfield.
 14. The method according to claim 13 wherein saidantigen-specific T cells are expanded according to the following method:(a) exposing said T cells to an anti-CD3 antibody which is immobilizedon a surface; and (b) stimulating an accessory molecule on the surfaceof the T cells with an anti-CD28 antibody, wherein said anti-CD28antibody is immobilized on the same surface as the anti-CD3 antibody;thereby inducing expansion of said antigen-specific T cells.
 15. Themethod according to claim 14, further comprising exposing said T cellsto IL-15.
 16. The method according to claim 14, further comprisingexposing said T cells to a natural ligand for CD137.
 17. The methodaccording to claim 14, further comprising exposing said T cells to ananti-CD137 antibody.
 18. The method according to claim 14, furthercomprising exposing said T cells to an anti-NKG2D antibody or a naturalligand for NKG2D. 19-20. (Canceled)
 21. The method according to claim 11wherein said antigen is selected from the group consisting of protein,glycoprotein, peptides, antibody/antigen complexes, whole tumor orvirus-infected cells, fixed tumor or virus-infected cells, heat-killedtumor or virus-infected cells, tumor lysate, non-soluble cell debris,apoptotic bodies, necrotic cells, whole tumor cells from a tumor or acell line that have been treated such that they are unable to continuedividing, allogeneic cells that have been treated such that they areunable to continue dividing, irradiated tumor cells, irradiatedallogeneic cells, natural or synthetic complex carbohydrates,lipoproteins, lipopolysaccharides, transformed cells or cell line,transfected cells or cell line, transduced cells or cell line, andvirally infected cells or cell line.
 22. The method according to claim11 wherein said antigen is attached to said surface by anantibody/ligand interaction.
 23. The method according to claim 22wherein said antibody/ligand interaction comprises an interactionbetween an antibody/ligand pair selected from the group consisting ofanti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1antibody /PR1, anti-PR3 antibody /PR3, anti-tyrosinaseantibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen,anti-MUC-1 antibody/MUC-1 antigen, anti-α-fetoproteinantibody/α-fetoprotein antigen, anti-Her2Neu antibody/Her2Neu, anti-HIVgp120 antibody/HIV gp120, anti-influenza HA antibody/influenza HA,anti-CMV pp65/CMV pp65, anti-hepatitis C antibody/hepatitis C proteins,anti-EBV EBNA 3B antibody/EBV EBNA 3B antigen, and anti-human Ig heavyand lignt chains/Ig from a myeloma cancer patient, and anti-human Igheavy and lignt chains/Ig from a CLL cancer patient.
 24. The methodaccording to claim 11 wherein said antigen is chemically attached tosaid surface.
 25. The method according to claim 11 wherein theattachment of said antigen to said surface comprises a biotin-avidininteraction.
 26. The method according to claim 11 wherein saidpopulation of cells wherein at least a portion thereof comprises APC isderived from a source selected from the group consisting ofleukapheresis product, peripheral blood, lymph node, tonsil, thymus,tissue biopsy, tumor, spleen, bone marrow, cord blood, CD34⁺ cells,monocytes, and adherent cells. 27-29. (Canceled)
 30. A population ofantigen-specific T cells generated according to the method of any one ofclaims
 1. 31. A composition comprising the antigen-specific T cellsaccording to claim 30 and a pharmaceutically acceptable excipient.
 32. Amethod for stimulating an immune response in a mammal comprising,administering to the mammal the composition of claim
 31. 33. A methodfor reducing the presence of cancer cells in a mammal comprising,exposing the cells to the composition of claim
 31. 34. The method ofclaim 33 wherein the cancer cells are from a cancer selected from thegroup consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's disease,leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer,prostate cancer, colo-rectal cancer, kidney cancer, renal cellcarcinoma, pancreatic cancer, esophageal cancer, brain cancer, lungcancer, ovarian cancer, cervical cancer, multiple myeloma, hepatoma,acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia(CLL).
 35. A method for inhibiting the development of a cancer in amammal, comprising administering to the mammal the composition of claim31.
 36. The method of claim 35 wherein the cancer cells are from acancer selected from the group consisting of melanoma, non-Hodgkin'slymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma,thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidneycancer, renal cell carcinoma, pancreatic cancer, esophageal cancer,brain cancer, lung cancer, ovarian cancer, cervical cancer, multiplemyeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), and chroniclymphocytic leukemia (CLL).
 37. A method for ameliorating an immuneresponse dysfunction in a mammal comprising administering to the mammalthe composition of claim
 31. 38. A method for reducing the presence ofan infectious organism in a mammal comprising, administering to themammal the composition of claim
 31. 39. The method of claim 38 whereinsaid organism is selected from the group consisting of a virus, asingle-stranded RNA virus, a single-stranded DNA virus, adouble-stranded DNA virus, Human Immunodeficiency Virus (HIV), HepatitisA, B, or C virus, Herpes Simplex Virus (HSV), Human Papilloma Virus(HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), a parasite, abacterium, M tuberculosis, Pneumocystis carinii, Candida, Aspergillus.40. A method for inhibiting the development of an infectious disease ina mammal, comprising administering to the mammal the composition ofclaim
 31. 41. The method of claim 40 wherein said organism is selectedfrom the group consisting of a virus, a single-stranded RNA virus, asingle-stranded DNA virus, a double-stranded DNA virus, HumanImmunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes SimplexVirus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus (CMV),Epstein-Barr virus (EBV), a parasite, a bacterium, M tuberculosis,Pneumocystis carinii, Candida, Aspergillus.